THE STUDY OF THE EFFECTS OF A CHANGE IN THE EXPRESSION OF MIXED LINEAGE LEUKEMIA 5 ON TRANSCRIPTION REGULATION LEE PEI BSc (Hons), National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 1 Acknowledgements I would like to express my utmost gratitude to my supervisor Dr Deng Lih-Wen for her guidance despite her other academic and professional commitments and her generous funding for the project. I would also like to thank my lab members, Yew Chow Wenn, Cheng Fei, Liu Jie for guiding me on the technical and analytical skills as wells as their encouragement and companionship all this while. I would like to offer special thanks to everyone who has helped me in one way or another in the course of my research project. I would also want to express my sincere thanks to the Department of Biochemistry for providing me the opportunity to do my research work. Lastly, I am grateful to my family for their constant encouragement and support throughout my graduate studies. 2 TABLE OF CONTENTS LIST OF FIGURES ················································································5 LIST OF TABLES ·················································································7 LIST OF ABBREVIATIONS ····································································8 LIST OF PUBLICATIONS ·····································································10 SUMMARY·························································································11 CHAPTER 1: INTROUDCTION 1.1 Nuclear speckles ················································································13 1.1.1 Discovery of nuclear speckles ···························································13 1.1.2 Characterization and dynamics of nuclear speckles ···································14 1.2 Splicing ··························································································15 1.2.1 An overview ···············································································15 1.3 Transcription ····················································································19 1.3.1 An overview ···············································································19 1.3.2 Coordination between transcription and splicing ······································20 1.3.3 Chromatin organization and transcription ··············································23 1.4 Mixed Lineage Leukemia (MLL) Protein Family ·········································24 1.4.1 A summary of MLL protein family ·····················································24 1.4.2 MLL protein family as human H3K4 specific methyltransferases ··················26 1.4.3 MLL protein family and transcription ··················································27 1.4.4 MLL protein family and pre-mRNA processing ······································29 1.5 Mixed Lineage Leukemia 5 (MLL5) ·······················································30 1.5.1 A summary of MLL5 ·····································································30 1.5.2 Current findings on MLL5 ·······························································31 1.5.2.1 MLL5 and cell cycle regulation ··················································31 1.5.2.2 MLL5 and DNA damage response ···············································31 1.5.2.3 MLL5 and animal studies ·························································32 1.5.2.4 MLL5 and epigenetic regulation ·················································33 1.6 Aims and objectives of the study ····························································34 CHAPTER 2: MATERIALS AND METHODS 2.1 Cell lines and culture conditions ·····························································37 2.2 RNA interference and delivery ······························································38 2.3 Cloning ··························································································40 2.4 Calcium-phosphate mediated DNA plasmid transfection·································42 2.5 Cell lysate preparation, Immunoprecipitation and Western blot ·························43 2.6 Immunofluorescence microscopy ···························································49 2.7 Nuclease digestion ·············································································49 3 2.8 RNA extraction, cDNA synthesis and semi -quantitative real-time PCR ··············50 2.9 Splicing assay ··················································································52 2.10 Bromo-uridine triphosphate incorporation in permeabilized cells ·····················55 2.11 Micrococcal nuclease (MNase) accessibility assay ······································55 CHAPTER 3: RESULTS 3.1 Co-localization of MLL5 with the spliceosome components ····························59 3.2 Localization of MLL5 and spliceosome components in response to nuclease and heat-shock treatment ············································································64 3.3 Association of MLL5 and SC35······························································67 3.4 Alteration in MLL5 protein level induced the redistribution of SC35 to enlarged speckle domains ················································································70 3.5 Multiple transcription inhibitors induce MLL5 to redistribute to enlarged speckles ··76 3.6 Intra-nuclear reorganization of MLL5 speckles is reversible and temperature dependent ························································································78 3.7 Alteration in MLL5 expression triggered transcription block ····························79 3.8 Association of MLL5 and RNAPII ··························································85 3.9 MLL5 overexpression resulted in a slower migration of Cyclin T1 ·····················87 3.10 MLL5 knockdown does not affect the phosphorylation state of RNAPII ·············89 3.11 MLL5 knockdown affects chromatin structure ···········································91 3.12 MLL5 and chromatin remodelling complex ··············································93 3.13 MLL5 and splicing activity ··································································95 CHATPER 4: DISCUSSION 4.1 An overview ····················································································98 4.2 Importance of maintaining MLL5 at a homeostatic level ································98 4.3 Plausible roles of MLL5 in transcription regulation ·····································105 4.3.1 MLL5 and its involvement in histone modifications································105 4.3.2 MLL5 and its involvement in chromatin organization······························107 CHAPTER 5: FUTURE DIRECTIONS AND CONCLUSION 5.1 Chromatin remodelling, histone modifications and DNA methylation – How does it all fit together? ·······························································109 5.2 Histone modifying properties of MLL5 – When does it occur? ·······················111 5.3 Cell cycle arrest or transcription inhibition – Which comes first? ····················112 5.4 Conclusion ····················································································113 REFERENCES ··················································································115 4 LIST OF FIGURES Figure 1: A simplified representation of the spliceosome assembly pathway and premRNA splicing …………………………………………………………...18 Figure 2: Integration of transcription and pre-mRNA processing…………………..21 Figure 3: Bi-directional coupling: a splicing factor regulates transcription, which in turn regulates alternative splicing ………………………………………..22 Figure 4: A schematic presentation of MLL family proteins……………………….26 Figure 5: Co-localization of MLL5 with the spliceosome components …………...60 Figure 6: Different anti-MLL5 antibodies and their co-localization with SC35 …..62 Figure 7: Co-localization of MLL5 with the spliceosome components in different cell lines ……………………………………………………………………....64 Figure 8: Association of MLL5 with splicing factor SC35 under RNase A digestion and heatshock ……………………………………………………………..67 Figure 9: Association of MLL5 with splicing factor SC35 …………………………69 Figure 10: SC35 protein expression remains unaltered in MLL5 depleted cells …..71 Figure 11: Alteration in MLL5 protein levels by RNA interference induced the redistribution of SC35 to enlarged speckle domains ……………………...73 Figure 12: Exogenous introduction of MLL5 induced the re-distribution of SC35 to enlarged speckle domains …………………………………………….....75 Figure 13: Multiple transcription inhibitors induce MLL5 to redistribute to enlarge Speckles…………………………………………………………………77 Figure 14: Re-distribution of MLL5 speckles is temperature dependent ……….....79 Figure 15: Gene expression of S14 ribosomal subunit after MLL5 knockdown ….80 Figure 16: Alteration in MLL5 expression by RNA interference triggers transcription block …………………………………………………………………....82 Figure 17: Exogenous introduction of MLL5 triggered transcription block ……...84 Figure 18: Distribution pattern of MLL5 and RNAPII …………………………….85 Figure 19: Association of MLL5 and RNAPII ……………………………………..87 5 Figure 20: MLL5 overexpression resulted in a slower migration of Cyclin T1…...89 Figure 21: MLL5 knockdown does not affect the phosphorylation state of RNAPII…………………………………………………………………..90 Figure 22: Analysis of chromatin modifications in MLL5 knockdown cells ……..92 Figure 23: Analysis of chromatin organization in MLL5 knockdown cells ……....93 Figure 24: Effect of MLL5 knockdown on SWI/SNF protein complex …………..94 Figure 25: A test system for determining the splicing efficiency in mammalian cells …………..………………………………………………………....96 Figure 26: Analysis of splicing efficiency in MLL5 knockdown cells ……………97 Figure 27: A model illustrating the participation of MLL5 in transcription and splicing processes ………………………………………………………104 Figure 28: Possible epigenetic modifications on the chromatin …………………..111 6 LIST OF TABLES Table 1: Nucleotide sequences of the siRNA used for MLL5 or SC35 gene Silencing ……………………………………………………………………39 Table 2: Optimised volumes as well as concentrations of Lipofectamine™ RNAiMAX (Invitrogen) and siRNAs used in preparation of the transfection mixes for MLL5 gene silencing ……………………………………………40 Table 3: PCR reaction composition and conditions of pXJ-HA-SC35 ………….....41 Table 4: Digestion reaction composition of pXJ-HA-SC35 ……………………......42 Table 5: Reaction composition for ligation of SC35 into pXJ-HA vector ………....42 Table 6: Transfection mixture using calcium-phosphate method for a typical 60mm dish …………………………………………………………………….......43 Table 7: Buffers used in Western Blot ……………………………………………...45 Table 8: Conditions for Western Blot ………………………………………………45 Table 9: Self-generated or commercial MLL5 antibodies used in Western blot, immunofluorescence and immunoprecipitation …...…………………......46 Table 10: Commercial antibodies and beads used in Western blot, immunofluorescence and immunoprecipitation ………………………....47 Table 11: cDNA synthesis conditions ……………………………………………..51 Table 12: Primers used in qPCR ……………………………………………….......51 Table 13: qPCR reaction mixture and conditions ...........................................…....52 Table 14: Preparation of media and reagents required for β-galactosidase activity activity ………………………………………………………………......54 Table 15: RT-PCR conditions ……………………………………………………...55 Table 16: Components of buffers used in MNase assay …………………………..58 7 LIST OF ABBREVATIONS Abbreviations ASCOM ASH2L ATCC BSA CBP CD CGBP ChIP CIP CT DAPI DMEM DRB FBS Br-UTP Gal H3 H4 HBS HCF HMT HOX HP1 HSC KD GO IGCs LAR II LT-HSC Luc MBD miRNA MLL5 Mnase NC-siRNA ONPG PcG PHD PF PML PS PS1 PS2 P-TEFb PTIP Full Names ASC-2-containing co-activator complexes Absent, Small or Homeotic-like American Type Culture Collection Bovine serum albumin CREB binding protein Central domain CpG-binding protein Chromatin immunoprecipitation Calf intestinal alkaline phosphatase C terminus 4’ 6-diamidino-2-phenylindole, dihydrochloride Dulbecco’s Modified Eagles Medium 5,6-dichlorobenzimidazole riboside Fetal bovine serum Bromo-uridine Triphosphate Galactosidase Histone 3 Histone 4 Hanks Buffered Salt Host cell factor Histone methyltransferase Homeobox Heterchromatin protein 1 Hematopoietic stem cells Knockdown Gene Ontology Interchromatin granule clusters Luciferase Assay Reagent II Long-term hematopoietic stem cells Luciferase Methyl-CpG-binding domain microRNA Mixed Lineage Leukemia 5 Micrococcal nuclease Negative control-siRNA o-Nitrophenyl-β-D-galactopyranoside Polycomb Plant homeodomain Perichromatin fibrils Promyelocytic leukaemia PHD SET Permeabilization solution 1 Permeabilization solution 2 Positive transcription elongation factor b Pax transactivation domain-interacting protein 8 qPCR RbBP5 RNA RNAPI RNAPII RNAPIIa RNAPIIo RNAPII CTD RT RT-PCR SC SET Sm snRNA snRNP SR SS SWI/SNF TrxG WB WDR5 Semi-quantitative polymerase chain reaction Retinoblastoma Binding protein 5 Ribonucleic acid RNA polymerase I RNA polymerase II Hypo-phosphorylated RNAPII Hyper-phosphorylated RNAPII RNA polymerase II C-terminal domain Room temperature Reverse transcription polymerase chain reaction Scrambled Su(var)3-9, enhancer-of-zeste and trithorax smith antigens small nuclear RNA Small nuclear ribonucleoproteins Serine / arginine Splice sites SWItch/Sucrose Non Fermentable Trithorax group Western blot WD Repeat Domain 5 9 LIST OF PUBLICATIONS Journal Articles 1. Yew CW, Lee P, Chan WK, Lim VK, Tay SK, Tan TM, Deng LW (2011). A Novel MLL5 Isoform That Is Essential to Activate E6 and E7 Transcription in HPV16/18-Associated Cervical Cancers. Cancer Res 2011 Nov 1;71(21):6696-707. 2. Lee P, Yew CW, Wu Q, Deng LW (2012) Impact of altering the basal level of Mixed Lineage Leukemia 5 on global chromatin organization and transcription regulation. (Manuscript to be submitted) 10 SUMMARY Mixed Lineage Leukaemia 5 (MLL5) is a mammalian Trithorax group (TrxG) gene located at chromosome band 7q22, a frequently deleted region in myeloid malignancies. MLL5 was discovered and subsequently cloned in year 2002. Currently, there are a total of fifteen publications dedicated to MLL5. MLL5 is identified as a nuclear protein and either over-expression or depletion of MLL5 resulted in dual-phase cell cycle arrest. In interphase cells, MLL5 exhibits distinct irregular, punctate intra-nuclear speckles but with uncharacterized biological functions. Intrigued by the complexities of nuclear speckles, which are dynamic structures enriched with a reservoir of factors that participate in transcription and premRNA processing, we attempted to unravel the biological functions of MLL5 within the nuclear speckles. To begin with, we examined the co-staining pattern of MLL5 with several well-characterized proteins that were known to display nuclear speckle pattern by immunofluorescence staining. Interestingly, we found that MLL5 nuclear speckles exhibited extensive co-localization with the spliceosome protein SC35 which has recently been reported to be involved in the bi-directional coupling of transcription and splicing. Given the fact that alterations in MLL5 level through ectopic over-expression or siRNA-mediated knockdown resulted in the enlargement and aggregation of nuclear speckles, a phenotype that indicated a defect in cotranscriptional splicing process, we therefore speculate a novel biological role of MLL5 involving in the transcription and splicing processes. We tested this hypothesis by examining if MLL5 is sensitive to transcription inhibitors and whether MLL5 is associated with RNA Polymerase II (RNAPII) transcription machinery. Results 11 showed that MLL5 not only physically interacted with RNAPII but also affected the progression of RNAPII along the DNA template as MLL5 depletion resulted in chromatin compaction and affected the subunits of chromatin remodelling proteins. In addition, histone signatures signifying transcription activation, namely H3K4 trimethylation and H4 acetylation, were largely reduced in MLL5-kockdown cells. Splicing activity was also reduced as a result of a disruption in the transcription process. Taken together, our findings suggest that MLL5 participates in transcription regulation, which consequently affects gene regulation and cell-cycle progression. 12 CHAPTER 1 – INTRODUCTION 1.1 Nuclear speckles 1.1.1 Discovery of nuclear speckles The pioneer work for nuclear speckles was reported by Santiago Ramo´n y Cajal in 1910 [reviewed in (Lafarga et al., 2009)]. In this study, Ramo´n used acid aniline stains to identify structures he described as “grumos hialinas”, which literally meant “translucent clumps”. In 1959, through the use of electron microscopy, Hewson Swift (Swift, 1959) observed particles in the cells to be localized in “clouds” instead of being randomly distributed. Further investigations by Swfit through cyto-chemical analysis suggested that these particles harboured ribonucleic acid (RNA). Swift termed these particles as interchromatin particles. It was only in 1961 when researcher J. Swason Beck (Beck, 1961), upon examining rat liver sections that were immunelabelled with serum from auto-immune disorder patients, coined the term “speckles” for the interchromatin particles that were discovered two years ago. However, it was only after several years later that the first connection between pre-mRNA splicing and nuclear speckles or interchromatin granules emerged. This was found through an examination of the distribution of small nuclear ribonucleoproteins (snRNP antigens) using anti-splicing factor-specific antibodies that illustrated a speckled distribution of snRNPs in the cell nuclei (Perraud et al., 1979; Lerner et al., 1981; Spector et al., 1983). These distinct classes of sub-nuclear bodies have always been an area of intense research even till present. 13 1.1.2 Characterization and dynamics of nuclear speckles The mammalian cell nucleus is a multi-functional and complex organelle where a plethora of cellular mechanisms occur in sub-nuclear compartments collectively termed as foci. These foci, approximately 20-50 of them diffusely distributed in the nucleoplasm, appeared as irregular, punctate structures with interconnections existing in variable shapes and sizes (Lamond and Spector, 2003). These distinct foci, identified as nuclear speckles and Cajal (coiled) bodies, are dynamic structures involved in transcription and pre-mRNA splicing (Spector, 1993; Matera, 1999). Further characterizations by electron microscopy revealed these nuclear speckles to co-localize in nuclear regions designated as interchromatin granules clusters (IGCs) and perichromatin fibres (PFs) (Fakan et al., 1984; Raska et al., 1990; Spector et al., 1993). Active pre-mRNA transcription pre-dominates at the PFs that are enriched with nascent DNA, RNA, RNA polymerase II (RNAPII) and histone modifiers for transcriptionally active chromatin. Splicing speckles observed in IGCs signifed the sites for splicing factor assembly and storage as well as the sites for splicing processes such as RNA editing and transport (Carter et al., 1991; Wang et al., 1991; Spector and Lamond, 2011). Nuclear speckles are dynamic structures and there is a continuous shuttling of splicing factors in and out of the speckles. In the event of transcription inhibition, either through the use of inhibitors or as a consequence of heat-shock, nuclear speckles became enlarged and rounded as splicing factors aggregate in them (Spector et al., 1991; Melcak et al., 2000). However, when the expression of intron-containing genes is high (Huang and Spector, 1996; Misteli et al., 1997) or during a viral infection 14 when transcription activity increases (Jimenez-Garcia and Spector, 1993; Bridge et al., 1995), the accumulation of splicing factors within the speckles decrease as they get distributed to the transcription sites in the nucleoplasm. Undeniably, much progress has been made in recent years towards a better understanding of the structure and function of the nuclear speckles. However, given the dynamic nature of the speckle morphology, answers to a number of questions remain. In particular, the detailed molecular mechanism on how the components of the nuclear speckles efficiently coordinate the complex events in the cell, how splicing factors systematically execute the splicing process, consequently giving rise to the different splice forms of the gene transcript. 1.2 Splicing 1.2.1 An overview Nuclear pre-mRNA splicing is an essential and important process that governs eukaryotic gene expression. It is a process where introns are excised and this occurs in the spliceosome complexes that constitute two different classes of snRNP antigens U1, U2, U4/U6, U5 (Bindereif and Green, 1990) and non-snRNP antigens like SC35 (Reed, 1990). Both groups belong to the serine/arginine (SR) family and share structural features including an RNA binding domain and a SR-rich domain that is responsible for their targeting to nuclear speckles (Zahler et al., 1992; Birney et al., 1993). These proteins function cooperatively to catalyse the excision of the intervening sequences in the pre-messenger RNA (pre-mRNA). 15 Among the SR protein family, SC35, discovered through a monoclonal antibody against partially purified spliceosomes, is commonly used to define splicing nuclear speckles (Fu and Maniatis, 1990). The group discovered that SC35 co-localized well with snRNPs within the speckled nuclear domains, thereby providing the first evidence that these speckled regions constituted both types of snRNPs. It has been reported that nuclear extracts depleted of SC35 was incapable of splicing exogenous pre-mRNA. However, this was a reversible process as splicing activity could be restored by complementing the extracts with SC35 antigen or other members of the SR family (Zahler et al., 1992). The process of pre-mRNA splicing constituted two trans-esterification reactions, namely lariat intron formation and exon ligation. Briefly, this occurred in an orderly step-wise manner, involving the interaction between the spliceosomal snRNPs and non-snRNPs such as splicing factors SC35. Briefly, U1-snRNP first associated with the 5' splice site, thereafter, the attachment of the U2-snRNP near the branch-point enable the entry of the U4/U5/U6 tri-snRNP complex to complete the spliceosome assembly. Structural rearrangements then occurred and this resulted in U1 and U4 expulsion, catalytic activation, lariat formation, exon ligation, spliced product release and the eventual association of the remaining components that constitute the spliceosome assembly. A simplified representation of the spliceosome assembly pathway and pre-mRNA splicing is illustrated in Figure 1. Over the years, extensive research has revealed that the splicing of pre-mRNA in eukaryotes is also tightly coupled to the transcription process and this occurs as nascent transcripts are synthesized from RNA polymerase II. In fact, unravelling the splicing process not only aid in having a better understanding of gene expression at the molecular level; 16 even at the medical level, it allows for better treatment and prognosis as aberrant premRNA splicing has been associated with the onset of human diseases. 17 Figure 1: A simplified representation of the spliceosome assembly pathway and pre-mRNA splicing. The pre-mRNA is depicted with rectangular boxes (blue) as exons, linked by a single intron (black line) from the 5’to the 3’ splice sites (SS). For simplicity, only the ordered interactions of the snRNPs (indicated by circles), but not those of non-snRNP proteins are illustrated. During the assembly phase, the spliceosomal snRNP U1 first assembles onto the pre-mRNA before the systematic recruitment of U2, followed by the other snRNPs. During activation, the Prp28associated complex joins the spliceosome while the U1 and U4 snRNPs depart. Catalysis proceeds in two steps: lariat formation and exon ligation. Eventually, the mRNA is released and the spliceosome is disassembled. Backward arrows indicate the reversibility of process as the cycle begins. [Adapted from (Will and Luhrmann, 2011)] 18 1.3 Transcription 1.3.1 An overview RNA polymerase II (RNAPII) is a key player in the transcription process. Prior to splicing, nascent RNA transcripts are generated by RNAPII. The RNAPII harbours 52 tandem consensus heptapeptide (YSPTSPS) repeats at its C-terminal domain (RNAPII CTD) (Corden, 1990) and phosphorylation on the multi-sites controls the state of transcription. RNAPII with un-phosphorylated CTD is recruited to the preinitiation site at the promoters while the transition between transcription initiation and elongation is mediated by multi-phosphorylation events that are catalysed by proteinkinase complexes. Cdk7-cyclinH phosphorylates RNAPII CTD at Serine-5, generating a hypo-phosphorylated RNAPII (RNAPIIa) that participates in transcriptional initiation. Phosphorylation at Serine-2 is catalysed by Cdk9-cyclinT, forming hyper-phosphorylated RNAPII (RNAPIIo) that associates with transcriptional elongation (Zawel et al., 1995). RNAPIIo has also been reported to exist in splicing factor-rich nuclear speckles (Bregman et al., 1995; Mortillaro et al., 1996) and significant enrichment and co-localization has been observed for Cyclin T1 with the nuclear speckles than Cdk9 (Herrmann and Mancini, 2001). A growing body of evidence has also suggested that Cdk9 not only regulates RNAPII activity, but also participates in co-transcriptional histone modifications and pre-mRNA processing like splicing and 3’ end processing (Pirngruber et al., 2009a; Pirngruber et al., 2009b). 19 1.3.2 Coordination between transcription and splicing Emerging evidence has proved that functional integration of transcription by RNAPII and RNA processing machineries are mutually beneficial for efficient and regulated gene expression. The transcription process progresses from the initiation phase to the elongation phase and finally, the termination phase and these coordinated events within the cell nucleus are briefly summarized in Figure 2. Research over the years has also suggested that RNAPII CTD is critical in coupling the transcription and splicing processes as several observations have associated the elongating RNAPII to pre-mRNA splicing (Corden and Patturajan, 1997; Bentley, 1999; Hirose and Manley, 2000). Phosphorylated CTD serves as a recruitment and docking site for mRNA processing factors (Greenleaf, 1993) and stimulates the early steps of spliceosome assembly (Hirose et al., 1999). Besides, the phosphorylated CTD also recruits chromatin modifiers such as histone methyltransferases Set 1/2 (Phatnani and Greenleaf, 2006; Yoh et al., 2008) and histone acetyltransferases p300 and PCAF (p300/CBP-associated factor) (Cho et al., 1998). Hence, the cycle of phosphorylation and de-phosphorylation at the CTD during each round of transcription may coordinate the recruitment of these processing factors at different states of mRNA formation. 20 Figure 2: Integration of transcription and pre-mRNA processing. RNAPII is modified on its CTD with Serine-5 phosphorylation predominately at the start of the gene (blue line) and Serine-2 phosphorylation in the middle and end of the gene (yellow line). 5’-Capping enzymes are recruited through direct interactions with Serine-5 phosphorylated CTD to catalyse the co-transcriptional capping reaction. Various splicing factors are recruited during the elongation phase of transcription to facilitate co-transcriptional splicing. These splicing factors are dependent on Serine-2 phosphorylation on the CTD. The 3’-end formation is functionally coupled to transcription termination. Importantly, increasing evidence now suggests that the transcription and RNA processing machineries are functionally integrated in a reciprocal fashion such that individual co-transcriptional processing events can influence transcription at different phases. [Adapted from (Pandit et al., 2008)]. Recently, Lin and colleagues (Caslini et al., 2009) has uncovered a new and important role in transcription for a splicing regulator protein, SC35, that has previously been thought to be involved primarily in the splicing process. In the study, SC35 is needed to promote RNAPII elongation in a subset of genes where depletion in SC35 dramatically caused a decrease in nascent RNA synthesized by RNAPII but has no effect on the transcription by RNA polymerase I. Through the use of chromatin 21 immunoprecipitation combined with microarrays (ChIP-chip), the group observed that RNAPII was accumulated within the gene body upon SC35 depletion, indicating RNAPII stalling before it reached the end of the gene. This stalling led to a decrease in RNAPII elongation, which was confirmed by measuring the nascent transcripts using a run-on assay that utilized non-radioactive nucleotides. In short, these findings confirm the involvement of SC35 in the bi-directional coupling between transcription and splicing. A schematic diagram of this bi-directional coupling is illustrated in Figure 3. Figure 3: Bi-directional coupling: a splicing factor regulates transcription, which in turn regulates alternative splicing. The splicing factor SC35 interacts with RNA polymerase II (Pol II) and the elongation factor P-TEFb and, via phosphorylation of the C-terminal domain (CTD) of Pol II at Serine2 (Ser2), stimulates transcriptional elongation. In parallel, high elongation rates allow the simultaneous presentation to the splicing machinery of strong and suboptimal 3’ splice sites, which favours the use of the stronger one, leading to skipping of an alternative exon. [Adapted from (Fededa and Kornblihtt, 2008)] 22 In summary, the continuous shuttling of splicing factors to active transcription sites brings the elongating and splicing complexes into close proximity to facilitate cotranscriptional splicing. Given the tight coupling of transcription with the downstream RNA processing steps, transcription inhibition may halt a chain of gene expression events and arrest complexes at various RNA metabolism stages. Such disruption in transcription activity causes nuclear speckles to accumulate in the cell nucleus in an aggregate manner. 1.3.3 Chromatin organization and transcription Extensive chromatin research over the years indicates that chromatin structure is a primary regulator of gene transcription. The dynamics of chromatin structure is tightly regulated through multiple mechanisms which include histone modifications, chromatin remodelling, histone variant incorporation and histone eviction. In this study, we will examine how histone modifications and chromatin remodelling affect transcription. Histone tails are susceptible to numerous post-translational modifications (Li et al., 2007). These modifications include methylation of arginine (R) residues; methylation, acetylation, ubiquitination, ADP-ribosylation, and sumoylation of lysines (K); and phosphorylation of serines and threonines. Among them, modifications pertaining to active transcription include acetylation of histone 3 and histone 4 (H3 and H4) or dior tri-methylation of H3K4; and these are classified as euchromatin modifications. Heterochromatin modifications are associated with inactive transcription, and methylation occurs on H3K9 or H3K27. These histone modifications consequently 23 cause a change in the net charge of the nucleosomes, which in turn could strengthen or weaken inter-or intranucleosomal DNA-histone interactions. These effects eventually affect RNAPII progression along the chromatin, thereby affecting transcription. Chromatin remodelling is an energy-dependent process which involves a transient unwrapping of DNA from histone octamers. This facilitates transcription factors to become accessible to nucleosomal DNA. An example of chromatin modellers are the SWItch/Sucrose Non-Fermentable (SWI/SNF) proteins, which are a group of highly conserved DNA-stimulated ATPase complex (Muchardt and Yaniv, 1999). Taken together, chromatin architecture and its dynamic nature has a crucial role in dictating the fate of DNA-related metabolic processes which include DNA repair/recombination/replication, in particular, transcription by RNAPII that will be highlighted in this thesis. 1.4 Mixed Lineage Leukemia (MLL) Protein Family 1.4.1 A summary of MLL protein family The mammalian mixed lineage leukemia (MLL) family comprises five members (MLL1, MLL2, MLL3, MLL4/ALR and MLL5) and these proteins are human homologues of the Drosophila Trithorax group (TrxG) gene. Vertebrate and Drosophila TrxG genes encode transcriptional regulators that are postulated to be involved in the maintenance of gene expression. Proteins that are encoded by TrxG repress Homeobox (HOX) gene expression while their other antagonistic parties, 24 polycomb group (PcG) proteins, maintain the HOX gene expression (Ziemin-van der Poel et al., 1991). The mechanisms by which these two evolutionally conserved genes maintain the HOX gene expressions occur at the epigenetic level by chromatin remodeling and histone modifications, upon the formation of multi-protein complexes (Muller et al., 2002; Schuettengruber et al., 2007). Since HOX gene expressions are essential in determining the fates of embryonic development and haematopoiesis, aberrant HOX gene expression may represent a major molecular consequence of leukaemia-associated genetic lesions (Orlando and Paro, 1995; Look, 1997; Dorrance et al., 2006) MLL protein family possesses variable number of cysteine-rich plant homeodomain (PHD), zinc fingers and a highly-conserved Su(var)3-9, enhancer-of-zeste and trithorax (SET) domain. A schematic representation of MLL protein family is illustrated in Figure 4. Structural and biochemical analysis show that PHD finger and SET domain are involved in protein-protein interactions (Gould, 1997; van Lohuizen, 1999). PHD finger is usually present in chromatin-associated proteins and has been reported to be associated with nucleosomes or specific nuclear protein partners (Aasland et al., 1995) or serve as binding or recognition modules for histone modifications (Mellor, 2006) while the SET domain possesses methyltransferase activity (Nakamura, et al. 2002). Among the MLL family, MLL1 is the most extensively studied. For instance, the existence of PHD fingers within MLL1 regulate homodimerization and are indispensable for the interaction with cyclophilin Cyp33 (Fair et al., 2001). 25 Figure 4: A schematic presentation of MLL family proteins. In comparison with other family members, MLL5 has a sole PHD finger and a centralized SET domain. The graph is constructed base on the domain analysis results from SMART (http://smart.embl-heidelberg.de/). The evolutionary relationship among the family members is drawn using cladogram from ClustalW (http://www.ebi.ac.uk/Tools/clustalw/) [Adapted from (Cheng et al., 2008) ] 1.4.2 MLL protein family as human H3K4 specific methyltransferases In human, there are at least eight H3K4-specific histone methyltransferases (HMTs) which include MLL protein family (MLL1, MLL2, MLL3, MLL4, MLL5, hSet1A, hSet1B and ASH1) (Dou et al., 2006). Members of the MLL protein family are the main epigenetic regulators of diverse gene types that are associated with cell-cycle regulation, embryogenesis and development. Within the MLL family, MLL1 is located on the human chromosome band 11q23 and has been the most extensively studied (Djabali et al., 1992; Gu et al., 1992). A study by Poet and colleagues (Ziemin-van der Poel et al., 1991) revealed that MLL1 is associated with chromosome translocations in myeloid and lymphoid leukemia. Similarly, Djabali and colleagues (Djabali et al., 1992) found that the recurring translocations on MLL1 resulted in infant and therapy-related leukemias. Closely 26 homologous to MLL1 is MLL2 where both share the same interacting partners (Liu et al., 2009). Findings by Hughes and Yokoyama groups (Hughes et al., 2004; Yokoyama et al., 2004) showed that both MLL1 and MLL2 formed H3K4 histone methyltransferase complexes that constituted WD Repeat Domain 5 (WDR5), Retinoblastoma Binding protein 5 (RbBP5) and Absent, Small or Homeotic-like (Drosophila) (ASH2L). In another study, human CpG-binding protein (CGBP) was found to interact with MLL1, MLL2 and human Set1, and was a core component of the HMT complexes (Ansari et al., 2008). Dou and colleagues (Dou et al., 2006) have successfully purified MLL1 complex that contained histone acetyl transferase, MOF and host cell factors (HCF1 and HCF2). On the other hand, MLL3 and MLL4 existed in ASC-2-containing co-activator complexes (ASCOM) (Goo et al., 2003; Lee et al., 2006) with their histone lysine methyltransferase activities often coupled to H3 acetylation and H3K27 demethylation (Lee et al., 2007; Nightingale et al., 2007). These independent studies suggested that MLL-associated HMT activity appeared to be functional only when they existed as multi-protein complexes and each MLLinteracting complex played a distinct role in regulating MLL-mediated histone methylation and gene activation. 1.4.3 MLL protein family and transcription Even though the members of MLL family are commonly associated with regulating the HOX genes and H3K4 methylation, recent studies have showed that MLL protein family participate in regulating the transcription of diverse gene types (Milne et al., 2005; Takeda et al., 2006; Caslini et al., 2009; Kim et al., 2009). In the work by Guenther and colleagues (Guenther et al., 2005) using a genome-wide promoter 27 binding assay, MLL1 and H3K4 tri-methylation was found to be enriched at the promoters of transcriptionally active genes, suggesting MLL1 as a positive global regulator of gene transcription. The group also discovered that MLL1 localized to microRNA (miRNA) loci that were associated with leukemia and haematopoiesis. Through a separate study utilizing gene expression profiling in murine cell lines (Mll+⁄+ and Mll-⁄-), Scharf and colleagues (Scharf et al., 2007) demonstrated that Mll1 was associated with both transcriptionally active and repressed genes. MLL1 was found to regulate other gene types that were involved in differentiation and organogenesis pathways (such as COL6A3, DCoH, gremlin, GDID4, GATA-6 and LIMK) and tumor suppressor proteins involved in cell cycle regulation (p27kip1 and GAS-1). MLL1 was also found to be associated with the gene expressions that were linked with leukemogenesis and other malignant transformations including HNF-3 ⁄ BF-1, Mlf1, FBJ, Tenascin C, PE31 ⁄TALLA-1 and tumor protein D52-like gene (Scharf et al., 2007). On the other hand, MLL3 and MLL4 functioned as a p53 co-activator and were needed for H3K4 tri-methylation and expression of endogenous p53 target genes, in the presence of the DNA-damaging agent, doxorubicin (Kim et al., 2009). The expression of p21, a downstream target gene of p53, was found to be significantly decreased in Mll3 deficient mice as compared to the wild-type mice. Even though the direct interaction of MLL3 and MLL4 with p53 resulted in transcription activation in vitro (Dou et al., 2005), both required the protein, Menin, that acted as a mediator before they could be successfully recruited to the promoter of p27 and p18 genes to regulate their gene expressions (Milne et al., 2005). Recently, it has also been reported that MLL1 depletion led to p53-dependent growth arrest (Caslini et al., 2009). 28 Recent findings have demonstrated MLL1 to be linked with the telomeres. MLL1 was reported to affect H3K4 methylation and transcription of telomere in a lengthdependent manner (Caslini et al., 2009). Studies showed that the depletion of MLL1 by RNA interference in human diploid fibroblasts caused telomere chromatin modification, telomere transcription and telomere capping, leading to the telomere damage response. In short, these observations suggested the diversified roles of MLL protein family in gene regulation apart from being a master regulator of the HOX gene. 1.4.4 MLL protein family and pre-mRNA processing Besides regulating the HOX genes, recent studies have suggested that MLL1 to MLL4 are involved in coordinating the transcription and splicing processes. ASC2 (a component of the ASCOM complex that contains MLL1 to MLL4) exhibited target gene specificity to MLL complexes and interacted with CoAA (a hnRNP-like protein) and CAPER, both of which were key components involved in the alternate splicing process (Auboeuf et al., 2005). In addition, MLL histone methylases, in particular, MLL2, MLL3 and MLL4, have been demonstrated to interact with nuclear receptor through critical involvement of ASCOM complex that interacted with players participating in alternative splicing. Besides, MLL complexes have also been reported to coordinate Ski-complex that was also an important component in mRNA splicing (Zhu et al., 2005). Even though these studies showed that MLL1 to MLL4 interacted either directly or indirectly with mRNA processing factors, the functional details of MLL1 to MLL4 in the mRNA processing events remains to be elucidated. 29 1.5 Mixed Lineage Leukemia 5 (MLL5) 1.5.1 A summary of MLL5 MLL5 gene was discovered in a search for candidate myeloid leukemia tumour suppressor genes from an estimated 2.5 Mb commonly deleted segment within chromosome band 7q22 (Emerling et al., 2002). MLL5 is the most recent identified member of the human Trithorax (Trx) family and comprises 1858 amino acids. MLL5 contains 25 exons and spans 73 kb of genomic DNA. It is homologous to Drosophilia CG9007 and is evolutionarily more distant to the other family member as shown in Figure 4 (Emerling et al., 2002). MLL5 is distantly related to the other family members evolutionally as it encodes only a single PHD domain instead of a cluster found in other members, with the SET domain located nearer to the N-terminal region of the protein. Recent studies have suggested that human MLL5 and mouse MLL5, as well as the murine paralog, Setd5, possess SET domains that have sequence homology to yeast SET3 and SET proteins (Glaser et al., 2006; Sun et al., 2008). In addition, it has also been suggested that MLL5 may be the functional homolog of the Saccharomyces cerevisiae SET3; MLL5 was discovered to be a component of the NCOR complex, which is postulated to be functionally similar to the SET3C complex (Lanz et al., 2006). In addition, unlike the other MLL family proteins, MLL5 lacks DNA binding motifs such as A-T hooks and the methyltransferase homology motifs, suggesting that MLL5 might not bind DNA but would instead modulate transcription indirectly via protein-protein interactions through the PHD and SET domains (Emerling et al., 2002; Deng et al., 2004). 30 1.5.2 Current findings on MLL5 1.5.2.1 MLL5 and cell cycle regulation It has been shown that ectopic over-expression of MLL5 inhibits cell cycle progression at G1 phase, a crucial DNA damage checkpoint that governs genomic stability (Deng et al., 2004). In addition, silencing of MLL5 gene expression by small interfering RNAs (siRNAs) retarded cell growth and reversibly arrested cells in G1 and G2/M phases (Cheng et al., 2008), possibly through the up-regulation of Cyclin Dependent Kinase (CDK) inhibitor p21 and the de-phosphorylation of the retinoblastoma protein (pRb). Upon MLL5 knockdown, the entry of quiescent myoblasts into S-phase was delayed, but the completion of S-phase progression was hastened (Sebastian et al., 2009). Genome-based RNA interference profiling in cell division has also revealed that MLL5 might function in cytokinesis and mitosis (Kittler et al., 2007). Recently, it has been demonstrated that the phosphorylation of MLL5 by mitotic kinase Cdc2 is crucial for mitotic entry (Liu et al., 2010). These findings suggest that MLL5 has different regulatory roles throughout cell cycle. 1.5.2.2 MLL5 and DNA damage response Apart from having a regulatory role in cell cycle progression, MLL5 has recently been shown to be involved in the DNA damage responses. MLL5 is involved in the camptothecin (CPT)-induced p53 activation (Cheng et al., 2011). The treatment of actively replicating cells with CPT led to the degradation of MLL5 protein in a timeand dose-dependent manner. The down-regulation of MLL5 resulted in the 31 phosphorylation of p53 at Ser392, which was abrogated by exogenous overexpression of MLL5. In MLL5-knockdown cells, p53 protein was stabilized and bound to DNA with higher affinity, consequently resulting in the activation of downstream genes. In short, MLL5 functions as a novel component in the regulation of p53 homeostasis and a new cellular determinant of CPT. 1.5.2.3 MLL5 and animal studies Recently, three independent studies, reporting the first genetic analysis of Mll5 deficiency in mice have been published (Heuser et al., 2009; Madan et al., 2009; Zhang et al., 2009). Zhang and colleagues created the mice by deleting exon 3 and 4 of Mll5 and discovered that Mll5-/- mice displayed postnatal lethality, retarded growth and a decrease of long-term hematopoietic stem cells (LT-HSC). However, these mice did not show an increase incidence of spontaneous tumours and no cell cycle defects in the stem cell compartments were detected. Madan and colleagues embarked a similar strategy and observed male sterility in addition to the observations made by Zhang’s group. Surviving Mll5-/- mice had reduced thymus, spleen and lymph node sizes. Unlike Zhang’s observations, Madan highlighted that Mll5 was needed to maintain the quiescent state of LT-HSC. Heuser and colleagues generated Mll5-/- mice by disrupting exon 3. It was found that apart from similar observations made by the previous groups, there was an increase incidence of eye infection in Mll5-/- mice as a consequence of defects in neutrophils maturation. Just like Zhang’s group, no mice developed spontaneous tumour growth. Recently, Yap and his colleagues demonstrated the consequences of MLL5 deficiency in the area of spermatogenesis and found that MLL5 has an important role in this process (Yap et al., 2011). Mll5 32 deficient mice experienced defects in terminal maturation and in the packaging of sperm. In addition, these sperm were observed to have malfunctions in their motility. Despite employing different strategies to create the Mll5 knockout mice, MLL5-/mice displayed postnatal lethality and retarded growth. In summary, these studies revealed that Mll5 plays a pivotal role in hematopoietic stem cell fitness and spermatogenesis but is dispensable for embryonic development. 1.5.2.4 MLL5 and epigenetic regulation By virtue of the SET domain, MLL1 to MLL4 possess Histone H3 Lysine 4 (H3K4)specific methyltransferase activity and play vital roles in gene activations and epigenetics. (Kuzin et al., 1994; Curradi et al., 2002). Therefore, there is a possibility that MLL5 may also possess intrinsic histone methyltransferase activity to regulate gene expression through chromatin remodelling. However, several reports suggested that MLL5 lacked such intrinsic methyltransferase activity (Nightingale et al., 2007; Madan et al., 2009). Sebastian and colleagues (Sebastian et al., 2009) demonstrated that although MLL5 lacks inherent histone methyltransferase activity, it is able to regulate the expression of histone modifying enzymes Lysine Specific Demethylase 1 (LSD1) and SET7/9 through an indirect mechanism. MLL5 has also be shown to induce quiescent myoblasts to regulate both cell cycle and differentiation through a hierarchy of chromatin and transcriptional regulators (Sebastian et al., 2009), suggesting that MLL5 may play an essential role in the novel chromatin regulatory mechanism. To date, it remains debatable if MLL5 possesses histone H3K4 methyltransferase (HKMT) activity. Nonetheless, a short N-terminal MLL5 isoform, MLL5α (609 amino acids), containing both PHD and SET domains was recently 33 found to act as a mono- and di-methyltransferase to H3K4 only after MLL5 has been glcNAcylated (Fujiki et al., 2009). This isoform was identified as part of a multisubunit complex, in association with nuclear retinoic acid receptor RARα and also facilitates retinoic acid-induced granulopoiesis. Another short N-terminal MLL5 isoform, MLL5β (503 amino acids), was found to have a critical role in activating E6/E7 gene transcription in HPV16/18-induced cervical through its interaction with transcription factor AP1 where AP1 binding site is located at the distal region of the HPV18 long control region (Yew et al., 2011). Interestingly, a recent report demonstrated the prognostic importance and the therapeutic potential of MLL5 in acute myeloid leukemia where high MLL5 expression is associated with high overall survival and relapse-free survival (Damm et al., 2011). In short, these findings have highlighted the multi-functional roles of MLL5 but the molecular details remain elusive. 1.6 Aims and objectives of the study To date, very little information is known about the specific interactions of MLL5 with the cellular machineries. The spatial organization of endogenous MLL5 in the cell has not been comprehensively elucidated. Functional characterisation by Deng et al (Deng et al., 2004) demonstrated that the MLL5 protein has at least three nuclear localisation signals and exhibited a speckled nuclear distribution with uncharacterized biological functions. The aim of my project is to delineate the functional significance of these MLL5 nuclear speckles. Our group has previously shown that the phosphorylation and cellular localization of MLL5 is cell-cycle dependent (Cheng et al., 2008; Liu et al., 2010). At interphase, MLL5 exhibited distinct intra-nuclear foci 34 (Deng et al., 2004). Phosphorylation by mitotic kinase Cdk1 resulted in the dissociation of MLL5 from condensed chromosome, causing the nuclear speckles to dissolve (Liu et al., 2010). When cells re-entered G1 cell phase, the intra-nuclear foci re-appeared. Since MLL5 participates in cell cycle regulation, we hypothesize that these dynamic and cell cycle-specific nuclear speckles may represent functional compartmentalization of nuclear processes such as DNA replication/repair, transcription or splicing. To begin with, I examined the co-staining pattern of MLL5 with several wellcharacterized proteins that were known to display nuclear speckle pattern by immunofluorescence staining and found that MLL5 co-localized with the splicing components, SC35 and the snRNP antigens. An alteration in the basal level of MLL5 resulted in an enlargement of nuclear speckle, a phenotype that is associated with premRNA splicing or transcription inhibition. These observations suggest the role of MLL5 in the transcription or splicing process. Given the close interplay between the transcription and splicing processes, the effects of changes in MLL5 expression level on transcription and splicing were examined. MLL5 formed aggregates and localized in enlarged nuclear speckles in respond to various transcription inhibitors. Br-UTP incorporation study revealed a drastic loss in transcription activity in both overexpression of MLL5 and MLL5-siRNA treated cells. Biochemical analyses demonstrated that MLL5 interacted with the transcription machinery complex, RNA polymerase II. MLL5 depletion resulted in chromatin compaction and affected the subunits of chromatin remodelling proteins. Collectively, these results suggest a novel cellular role of MLL5 in transcription regulation, thereby contributing to gene regulation and cell cycle progression. Maintaining a proper intracellular balance of 35 MLL5 will also be important in providing a framework for proper cellular development as marginal alterations could serve as a determinant for the onset of diseases. Most importantly, elucidating the transcriptional and splicing regulation not only enable us to advance the knowledge of multilevel gene regulation in cells under physiological conditions but also provide opportunities to improve potential clinical therapies since genes are functionally organized into pathways. 36 CHAPTER 2 – MATERIALS AND METHODS 2.1 Cell lines and culture conditions Human cervical carcinoma HeLa, embryonic kidney cells HEK 293T, osteosarcoma U2OS, human colorectal carcinoma HCT116, human diploid fibroblasts WI38 and African green monkey kidney fibroblast-like cell line COS7 were cultured as monolayer in Dulbecco’s Modified Eagles Medium (DMEM, Gibco) in 25 cm2 tissue culture flasks. The cells were routinely passaged at 1:6 ratios (v/v) thrice weekly with the use of 1.0 ml of 0.25 % Trypsin-Ethylene-Diamine Tetracetic acid (EDTA) (GIBCO®). All cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). For WI-38 cell line, cells with less than 10 passages were used for the experiments. The media was supplemented with 10% fetal bovine serum (FBS, Hyclone), L-glutamine (2mM) (Gibco), penicillin (100 units/ml) and streptomycin (100 µg/ml) at 37°C with 5 % CO2. This medium will be referred as complete medium in subsequent experiment. Transcriptional inhibitors were added to the complete media at the indicated final concentrations and duration: α-amanitin (10 µg/ml, 8 h) (CalBioChem #129741); 5,6-dichlorobenzimidazole riboside (DRB, 100 µM, 3 h) (CalBioChem #D1916); Actinomycin D (20 µg/ml, 2 h) (Sigma #A9415); Roscovitine (25µM, 1.5hr) (Sigma #R7772). 37 2.2 RNA interference and delivery BLOCK iTTM RNAi designer software (Invitrogen, Carlsbad, CA, USA) were used to identify potential siRNA targeting sites within human MLL5 mRNA sequence. Three different MLL5 specific siRNA duplexes (#1, #2 and #3) targeting nucleotide positions at 1063, 5215 and 6807 respectively, from the transcription starting point [National Centre for Biotechnology Information (NCBI) reference sequence: NM_182931.2]. Two different SC35 specific siRNA duplexes (#1 and #2) were designed to specifically target human SC35 mRNA sequence at nucleotide positions 346 and 427 respectively from the transcription starting point [National Centre for Biotechnology Information (NCBI) reference sequence: NM_003016.4]. SC35 siRNA #2 was from Invitrogen (Stealth Select RNAi, SFRS2, Invitrogen). Scrambled siRNA was used as a control. All the siRNA duplexes were synthesized by 1st BASE (Singapore) and the sequences are summarized in Table 1. Cells were seeded one day before to achieve cell confluency of 40-60 % on the day of transfection. In performing siRNA transfection, cells were cultured in complete media. Transfection mixtures consist of Lipofectamine™ RNAiMAX (Invitrogen™) and siRNA were diluted with serum-free DMEM. The specific quantities of the reagent and siRNA added in preparation of the transfection mixes for the different cell culture vessels are summarised in Table 2. The transfection mix was incubated at room temperature (RT) for approximately 20 min to allow for the formation of siRNA duplex-Lipofectamine™ RNAiMAX complexes, before adding drop-wise into the cell culture vessels. To enhance the knockdown efficiency using MLL5 siRNA #2 and #3, as well as to achieve a knockdown efficiency that was comparable to MLL5 38 siRNA #1, a second transfection was carried out 24 h after the first. The cell media was subsequently changed 24 h post-transfection. Cells were cultured for 72 h posttransfection, following which the cells were harvested for the necessary assays and experiments. Transfection efficiencies were analysed by Western Blot. Table 1: Nucleotide sequences of the siRNA used for MLL5 or SC35 gene silencing siRNA ID siRNA sequences NC (Scrambled) Sense 5’-UUCUCCGAACGUGUCACGUdTdT-3’ Antisense 5’-ACGUCACACGUUCGGAGAAdTdT-3’ Sense 5’-CGCCGGAAAAGGGAAAAUAdTdT-3’ Antisense 5’-UAUUUUCCCUUUUCCGGCGdTdT-3’ Sense 5’- CAGCCCUCUGCAAACUUUCAGAAUUdTdT-3’ Antisense 5’-AAUUCUGAAAGUUUGCAGAGGGCUGdTdT3’ Sense 5’-GCACUG GUUGGGCAUUUUAdTdT-3’ Antisense 5’-UAAAAUGCCCAACCAGUGCdTdT-3’ Sense 5’-GCGUCUUCGAGAAGUACGGdTdT-3’ Antisense 5’-CCGUACUUCUCGAAGACGCdTdT-3’ Sense 5’-UCGUUCGCUUUCACGACAAdTdT-3’; Antisense 5’-UUGUCGUGAAAGCGAACGAdTdT-3’ MLL5 #1 (1063) MLL5 #2 (5215) MLL5 #3 (6807) SC35 #1 (346) SC35 #2 (427) 39 Table 2: Optimised volumes as well as concentrations of Lipofectamine™ RNAiMAX (Invitrogen) and siRNAs used in preparation of the transfection mixes for MLL5 gene silencing (Adapted: Invitrogen™ User Manual). Cell culture vessel Relative Surface Area Amount of siRNA (pmol) in serum-free DMEM(μl) Volume of Lipofectamine ™ RNAiMAX (μl) in serumfree DMEM(μl) Total Final siRNA volume of concentration antibiotics- (nM) free plating medium (ml) 1 12 in100 1.6 in 100 1.0 12 2.5 24 in 200 3.2 in 200 2.0 12 5.5 64 in 500 8.5 in 500 5.0 12 12-well plate 6-well plate 60mm plate 2.3 Cloning Full length MLL5 and MLL5 deletion mutants used in this study were generated by flanking each PCR fragment of MLL5 cDNA with the FLAG sequence and cloning the fragments into the pEF6/V5-His-vector (Invitrogen) in frame with BamHI and XbaI sites (Liu et al., 2010). SC35 cDNA was amplified by PCR from total RNA prepared from HeLa cells using the forward primer CGCGGATCCATGAGCTACGGCCGCCCCCCTCCCGATGT-3’ 5’(with BamHI cutting site) and reverse primer 5’-CCGCTCGAGTTAAGAGGACACCGCTCCTT- 40 3’ (with XhoI cutting site) and cloned in-frame into the pXJ40-HA vector with the conditions listed in Table 3. The PCR reaction was analysed by gel electrophoresis and the PCR product (666 bp) was purified directly from the PCR reaction mix using the PCR Purification Kit (Qiagen). DNA was eluted in 50µl of elution buffer (10mM Tris-Cl, pH 8.5). All the restriction enzymes (RE) used were purchased from New England Biolabs and the digestion reaction is summarised in Table 4. Ligation of the SC35 amplicon into the pXJ-HA vector was performed using T4 DNA ligation mixture (New England Biolabs). A total of 15µl ligation reaction was set up as shown in Table 5 and a negative control that consisted only the pXJ-HA vector was included. A 3:1 (vector: insert) ratio was used in the ligation process and the ligation mixture was incubated at 16°C overnight. The final construct, pXJ-HA-SC35, was verified by DNA sequencing. Subsequent sequences obtained were aligned against the relevant GenBank sequence using the Basic Alignment Search Tool (BLAST) from NCBI and in-frame fusions were also checked. Table 3: PCR reaction composition and conditions of pXJ-HA-SC35 PCR reaction mix Reagent 10X High Fidelity PCR buffer 2 mM dNTP 50 mM MgSO4 Primer Mix (10 µM each) Reverse primer Forward primer Template (HeLa cDNA)(100ng/µl) Platinum® Taq High Fidelity Autoclaved distilled water Total Quantity (µl) 5.0 PCR conditions 1) Initial denaturation: 94°C for 3 min 1.0 2.0 2) 35 cycles of a) DNA Denaturation: 94°C for 30 sec b) Primer annealing: 56.9 °C for 1 min c) DNA Extension: 68°C for 45 sec 2.0 2.0 1.0 3) Final extension: 72°C for 10mins 4) After cycling, the reaction is maintained at 18°C. 0.2 36.8 50.0 41 Table 4: Digestion reaction composition of pXJ-HA-SC35 Component Insert /Vector BamHI XhoI 10X NEB buffer 3 Bovine Serum Albumin (BSA) (10X) Water Calf Intestinal alkaline phosphatase (CIP) Total Insert (SC35) 30 µl 1µl 1µl 5µl 5µl 8µl 50µl Vector (pXJ40-HA) 30 (100ng/µl) 1µl 1µl 5µl 5µl 6.5µl 1.5µl 50µl Table 5: Reaction composition for ligation of SC35 into pXJ-HA vector Reagent 10X T4 DNA ligase buffer T4 DNA ligase pXJ-HA vector (16ng/µl) SC35 insert (44ng/µl) Water Total Ligation reaction (µl) 1.5 0.5 2.0 0.5 10 15 Negative control (µl) 1.5 0.5 2.0 10.5 15 2.4 Calcium-phosphate mediated DNA plasmid transfection 293T cells were seeded on 60 mm plate to achieve approximately 50% cell confluency on the day of transfection. Calcium-phosphate method was used for introducing DNA into the cells. The transfection mixture for a typical 60mm dish is listed in Table 6. To a 1.5ml eppendof tube, DNA was added to the middle part of the water while CaCl2 was added to the bottom part of the water. This DNA-CaCl2 mixture was mixed gently and thoroughly before transferred drop-wise to another 1.5ml eppendof tube containing 2X HBS solution. This DNA-CaCl2–HBS mixture was mixed gently with the pipette till the solution is homogenous and this transfection mixture was incubated at room temperature for 30 min before adding drop-wise 42 slowly into the cell culture vessel. After 24 h, fresh medium was given to the cells. Transfection efficiency was analyzed by Western Blot. Table 6: Transfection mixture using calcium-phosphate method for a typical 60mm dish Components Volume (µl) DNA (100ng/ µl) Variable (3 to 6µg DNA) 2.5M Calcium chloride solution 22 Water Variable Add the DNA-calcium chloride mixture drop-wise into 2X HBS solution 2X Hanks Buffered Salt Solution 230 (HBS) 2.5 Cell lysate preparation, Immunoprecipitation and Western blot Total cellular protein extraction was performed by direct cell lysis using Laemmli sample buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 10% glycerol, 0.01% bromophenol blue), boiled at 100°C for 3 min and sonicated for 20 sec at 30% output power when necessary (Sonics VCX130, Newtown, CT, USA). Cell lysates were made to a concentration of 20 million cells/ml. The buffers and conditions used for Western Blot can be found in Table 7 and Table 8 respectively. MLL5 protein is detected using either self-generated or commercially available atni-MLL5 antibodies listed in Table 9 while other proteins of interest are detected using commercial antibodies listed in Table 10. 43 For immunoprecipitation studies, cells were lysed in lysis buffer supplemented with protease and phosphatase inhibitors (150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM Na3VO4, and 5 mM NaF). In order to avoid protein degradation resulting from the inactivation of protease and phosphatase inhibitors, all subsequent steps involving the handling of the cell lysate were performed at low temperature on ice, where possible. The lysate were repeatedly passaged through a syringe needle (1 ml syringe with a 21 gauge size needle) to shear DNA and thus release the nuclear proteins. The lysates were then incubated on ice for 30 mins before centrifugation at 13000rpm for 15 mins at 4oC. The pellet was discarded and supernatant retained. 10 μl of the cell lysate was used to test for transfection efficiency before the remaining cell lysate was subjected to immunoprecipitation. A pre-clearing step to remove non-specific binding was performed by incubating the cell lysate with 20 μl TrueBlot™ Anti-mouse / rabbit Immunoglobulin Immunoprecipitation (IP) beads (50% slurry) at 4oC for 1 h with continuous rotation. Pre-cleared lysate were then divided into two portions before incubation with antibodies or IgG (Table 9 and Table 10) respectively at 4oC for 2.5 h with continuous rotation. All steps were done on ice. IP Beads was washed twice with ice-cold 1X PBS before incubation with each of the cell lysate-antibody mixtures and rotated at 4oC for another 1.5 h. Following that, IP beads were spun down at 1000g for 5 mins. The supernatant were kept as flow-through at -80oC, in the event that pulldown was not successful. The beads were washed once with ice-cold mild lysis buffer and twice with ice-cold 1X PBS. Proteins bound to the beads were then eluted with 60 μl of Sodium Dodecyl Sulphate (SDS)/Dithiothreitol (DTT) (4:1 ratio) and boiled at 100oC for 3 mins. The beads were then spun down at 13500 rpm for 2 mins to 44 dissociate the bound proteins. The supernatant was kept and analysis of Co-IP was performed by Western Blot. Table 7: Buffers used in Western Blot SDS-PAGE running buffer Transfer (protein KDa) < Buffer Transfer Buffer 150 (protein ≥ 150 TBS KDa) 25 mM Tris base 100 mM Tris base 25 mM Tris base 150 mM glycine 0.384 M glycine 150 mM glycine 0.1% SDS 20% (v/v) methanol 20% methanol (v/v) 0.05% SDS 10 mM HCl, Tris- 150 mM NaCl, 2.5 mM KCl (adjust to pH7.5) Table 8: Conditions for Western Blot Antibodies MLL5 antibody FLAG M2 antibody SantaCruz antibodies Primary Blocking buffer antibody diluent 5% skim milk 5% skim milk (Fluka*) in TBS (Fluka*) in TBS Secondary Washing antibody diluent 5% skim milk TBS/0.05% (Fluka*) in TBS Triton X-100 5% milk 5% milk † † (Anlene ) inTBS (Anlene ) in TBS 5% skim milk 5% skim milk * (Fluka ) in TBS (Fluka*) in TBS 5% milk (Anlene†) in TBS 5% skim milk (Fluka*) in TBS TBS/0.05% Tween-20 5% skim milk (Fluka*) in TBS/0.05% Tween-20 5% skim milk (Fluka*) in TBS/0.05% Tween-20 TBS/0.05% Tween-20 Upstate /BD Bioscience/ Abcam/ Cell Signalling Covance antibodies antibodies 5% skim milk 5% skim milk * (Fluka ) in (Fluka*) in TBS/0.05% TBS/0.05% Tween-20 Tween-20 5% BSA in 5% BSA in TBS/0.05% TBS/0.05% Tween-20 Tween-20 * Fluka skim milk, #70166; † Anlene Gold Hi-Calcium Skimmed milk. 45 TBS/0.05% Tween-20 TBS/0.05% Tween-20 Table 9: Self-generated or commercial MLL5 antibodies used in Western blot, immunofluorescence and immunoprecipitation. Antibodies or Beads Manufacturer Amino acid residues number of full length- Catalogue MLL5 No. Dilution Factor IF IP WB MLL5-8009* Alpha Diagnostic 1157-1170 - 1:100 15µg 1:5000 MLL5-227* Alpha Diagnostic 227-241 - 1:100 - - MLL5 Abcam 75339 Abcam Synthetic peptide derived from the N terminal 75339 1:100 - - MLL5 Abgent 6186a Abgent Not disclosed on the product sheet 6186a 1:50 - - MLL5 Orbigen 10849 Orbigen Not disclosed on the product sheet 10849 1:50 - - MLL5 Santa Cruz L14 Santa Cruz Epitope mapping near the N-terminus of human 68635 1:50 - - Santa Cruz MLL5 Epitope mapping near the N-terminus of human 68635 1:50 - - MLL5 Santa Cruz N20 * MLL5 Polyclonal antibody against human MLL5 central region was raised in rabbits and purified using a Protein A column (GE Healthcare, Piscataway, NJ, USA) (Liu et al., 2010). IF: Immunofluorescence IP: Immunoprecipitation WB: Western Blot 46 Table 10: Commercial antibodies and beads used in Western blot, immunofluorescence and immunoprecipitation. Antibodies or Beads Manufacturer Catalogue No. Dilution Factor IF IP WB PML SantaCruz sc-966 1:50 - - CBP SantaCruz sc-7300 1:50 - - CREST ImmunoVision HCT 0100 1:250 - - SC35 Sigma-Aldrich S4045 1:2000 - - SC35 BD Bioscience 556363 - - 1:200 Sm BD Bioscience MS-450-P1 1:500 - - CDK9 SantaCruz sc-13130 - - 1:200 CTD4H8 Millipore 05-623 1:1000 - 1:3000 H5 Covance MMS-129R 1:50 - 1:250 H14 Covance MMS-134R 1:50 - 1:250 8WG16 Covance MMS-126R 1:50 - 1:250 Cyclin T1 SantaCruz sc-8127 - 5µg 1:200 H3K4-3Me Abcam Ab1012 1:100 - 1:2000 H3K4-2Me Abcam Ab32356 - - 1:1000 H3K4-1Me Abcam Ab8895-25 - - 1:1000 H3K9-3Me Abcam Ab8898 1:250 - 1:1000 47 Histone H3 Cell Signalling 9715 - - 1:1000 Acetyl Histone H4 Upstate 06-866 1:250 - 1:2000 Brm Santa Cruz - - 1:200 Brg1 Santa Cruz - - 1:500 Baf155 Santa Cruz - - 1:200 FLAG M2 Sigma-Aldrich F3165 1:1000 - 1:2000 HA SantaCruz sc-805 - - 1:500 Tubulin SantaCruz sc-8035 - - 1:500 Goat anti-mouse HRP-conjugated GE Healthcare RPN-4201 - - 1:10000 Donkey anti-rabbit HRP conjugated Pierce (Thermo) 31238 - - 1:5000 anti-Mouse, F(ab’)2 HRP-conjugated Jackson 115-036-006 - - 1:5000 anti-GFP, IgG, Alexa Fluor 488 conjugate Invitrogen ImmunoResearch 21311 1:250 - 1:2000 anti-BrdU Alexa Fluor 594-conjugated Invitrogen A21304 1:250 - - Goat anti-mouse Alexa Fluor 568 -conjugated Invitrogen A11031 1:250 - - Chicken anti-rabbit Alexa Fluor 488 - Invitrogen A11008 1:250 - - Goat anti-human Alexa Fluor 594-conjugated conjugated Mouse IgG Invitrogen A11014 1:250 - - SantaCruz sc-2025 5µg - - Mouse IgG Mouse beads (50% slurry) eBioscience #00-8811 - 20µl 48 2.6 Immunofluorescence microscopy Cells were grown on poly-D-lysine (1mg/ml) (Sigma, Cat No. 6403) coated coverslips and fixed with methanol at -20°C for 10 min, rehydrated with 1X PBS and blocked in 5% bovine serum albumin. Respective primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. Samples were washed with PBS/0.05% Tween 20 thrice and incubated with secondary antibodies conjugated with Alexa Fluor 488 (green) or 568/594 (red) (Invitrogen) for 1 h. Antibodies used can be found in Table 9 and Table 10. DNA was stained with 4’ 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Invitrogen #D1306) and the coverslips were mounted with FluorSave reagent (Merck #345789) to preserve fluorescence. When necessary, at least 100 cells were counted for each sample. Images were acquired Olympus IX81 microscope equipped with a cooled charge-coupled device camera (QImaging) and analyzed using QEDInVitro™ Version 3.2.2 and Image-Pro Plus 6.2 software (MediaCybernetics). 2.7 Nuclease digestion Cells were fixed in methanol for 10 min at -20°C, rinsed in 1X PBS and incubated in RNase A (100 µg/ml in PBS, DNase free) (Sigma) for 2 h at 25°C. After several washes with 1X PBS, cells were prepared for immunofluorescence microscopy as described above. Heat-shock experiment was done in petri dishes containing coverslips and pre-warmed medium was incubated in a 45°C oven for 15 min prior to fixation for immunofluorescence microscopy. Control cells were also transferred to dishes and incubated for the same duration in medium kept at 37°C. 49 2.8 RNA extraction, cDNA synthesis and semi-quantitative real-time PCR (qPCR) Total RNA was extracted using TRIzol reagent (Invitrogen #15596-026) and the cDNA was synthesized using iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). About 1 million cells were collected by trypsinization, followed by centrifugation at 200 x g for 3 min at 4°C. Cell pellet was homogenized in 1 ml TRIzol reagent for 5 min at room temperature. Chloroform (200 µl) was added to the homogenized sample and mixed vigorously for 15 sec. After incubation for 5 min at room temperature and centrifugation at 13 000rpm for 15 min at 4°C, the upper aqueous phase (450 µl) was transferred to a new RNase-free eppendorf tube. RNA was precipitated by addition of 0.5 ml isopropanol and collected by centrifugation at 13 000rpm for 10 min at 4°C. The RNA pellet was washed with 75% ethanol (prepared using absolute ethanol and nuclease-free water), briefly air-dried, and dissolved in nuclease-free H2O (Ambion, #AM9939). The RNA concentration was determined by measurement of absorbance at 260 nm using NanoDrop 2000c (Wilmington, DE, USA). For cDNA synthesis, RNA that was extracted from cells was converted to cDNA using the cDNA synthesis kit with random hexamer primers (Biorad) and iCycler (Bio-Rad) machine. The reaction mix (20 µl) and the conditions were set up as shown in Table 11. 50 Table 11: cDNA synthesis conditions cDNA reaction mix Reagent iScript reaction mix iScript reverse transcriptase Random hexamers RNA (up to 1.5 µg) Nuclease free water Total Quantity (µl) 4.0 1.0 cDNA conditions 1) 5 min at 25°C, 2) 30 min at 42°C 3) 5 min at 85°C 1.0 Variable Variable 20.0 For semi-quantitative real time PCR (qPCR), KAPA SYBR FAST One-step qPCR Master mix is used. The various gene expression levels were measured using the iQ5 qPCR machine (Biorad) and in-house designed primers (Table 12). The reaction mix (50 µl for triplicates of each gene to be studied) and qPCR conditions are summarized in Table 13. Table 12: Primers used in qPCR Primers Sequence MLL5 Sense 5’ - CCA CCA CAA AAG AAA AAG GTT TCT C -3’ Antisense 5’- GTG TTG GTA AAG GTA GGC TAG C – 3’ Sense 5’-GTG AAG GTC GGA GTC AAC G-3’ Antisense 5’ TGA GGT CAA TGA AGG GGT C -3’ Sense 5’- GTT TTG CTT CAG GGA GGA GCT T-3’ Antisense 5’-AAC AAA CGA GAT TAG CGT GGG -3’ GAPDH S14 51 Table 13: qPCR reaction mixture and conditions qPCR reaction mix Reagent KAPA SYBR® FAST qPCR Master Mix (2X) Forward Primer (10 μM) Reverse Primer (10 μM) RNA (100ng) KAPA RT Mix (50X) Nuclease free water Total Quantity (µl) 25 qPCR conditions 1) Inactivate reverse transcriptase: 95°C for 5 min 2) PCR cycling and detection - 40 cycles a) Denaturation: 95°C for 3 sec b) Annealing / Extension: 60°C for 20 sec (data acquisition step) 1.0 1.0 Variable 1.0 Variable 50.0 3) Melt curve analysis 95°C for 1 min 55°C for 1 min 55°C for 10 sec (80 cycles, increasing each by 0.5°C each cycle) 2.9 Splicing assay Splicing efficiency assays were performed as described by (Nasim et al., 2002). Briefly, cells were seeded in a 6 well-plate and MLL5 knockdown was done the next day after cell plating. 24 h after knockdown, pTN23 plasmid was transfected into 293T using calcium phosphate method as described in Section 2.4. To ensure that MLL5 level remained minimal in the cell, knockdown was done again 24 h after transfection. In all, MLL5 knockdown was done for 72 h and over-expression of pTN23 was done for 48 h. After which, cells were harvested from each well of the 6well plate and the final cell pellet was re-suspended in 1 ml 1X PBS. 3/5 of the cell suspension was used for RNA extraction, 1/5 was kept for Western blot to check for MLL5 knockdown efficiency and the remaining 1/5 of the cell suspension was used for measuring β-galactosidase (Genomax) and luciferase activities (Promega). 52 To measure luciferase activities, cell pellet was lyzed in 150 µl of 1X passive lysis buffer for 30 min at room temperature with gentle rocking on an orbital shaker. After that, the lysates were transferred to a tube and centrifuged at 1000 rpm for 5 min. The supernatant (around 120 µl) was equally distributed for luciferase and β-galactosidase activities. Triplicates of each assay were done in a 96-well flat bottom micro-titer dish and 20 µl of the supernatant was used each time. 100 µl of Luciferase Assay Reagent (LAR II) was dispensed into each well and mixed by pipetting 3 times before placing the dish in the luminometer (Tecan) to measure the luciferase reading. The luminometer was programmed to perform a 2-second pre-measurement delay, followed by a 10-second measurement period for each reporter assay. Normalization of luciferase reading by β-galactosidase reading was carried out before comparisons were made. To measure β-galactosidase activity, 20 µl of cell lysate was added into a 96-well flat bottom micro-titer dish. For each well, 140 µl of Buffer A- β-mercaptoethanol was added and this buffer was prepared in the following way: 160 µl Buffer A-βmercaptoethanol mixture constituted 8 µl of 1M β-mercaptoethanol and 152 µl Buffer A. Mix the two components by inversion. The final volume in each well was 160 µl. The micro-titer dish was covered and incubated for 5 min at 37°C. After which, 50 μl of o-Nitrophenyl-β-D-galactopyranoside (ONPG) substrate was added to each well and the micro-titer dish was covered with a micro-titer dish lid. The dish was then incubated in an incubator at 37°C until the mixture turned bright yellow. To terminate the reaction, 90 μl of stop solution was added and the micro-titer dish was scanned in a micro-titer dish reader that was set at 415nm. The incubation period was recorded and this was the time expired between the addition of ONPG substrate and the 53 addition of the stop solution. The optimal OD415 reading is between 0.6 to 0.9. Table 14 summarized the preparation of media and reagents required for β-galactosidase activity. Table 14: Preparation of media and reagents required for β-galactosidase activity Reagents Components Buffer A–β-Mercaptoethanol Mixture (pH Buffer A 100 mM NaH2PO4 7.5) 10 mM KCl 1 mM MgSO4 Prepare fresh before each assay. 50 mM β-Mercaptoethanol o-Nitrophenyl-β-D-Galactopyranoside 4 mg/ml in 100 mM NaH2PO4 buffer (pH 7.5) (ONPG) Stop solution 1 M Na2CO3 To determine the splicing efficiency through reverse transcription polymerase chain reaction (RT-PCR), cell lysates were harvested and RNA extraction and cDNA synthesis was performed as described in Section 2.8. A 50 µl reaction mix was set up as shown in Table 15 and the primers used are as follows: GalF 3301.forward (5’AACATCAGCCGCTACAGTCAA-3’) and LucR 3700 (5’-ACGTGATGT TCTCCTCGATAT-3’). The final PCR products were visualised on a 2.5% agarose gel. 54 Table 15: RT-PCR conditions PCR reaction mix Reagent 10X High Fidelity PCR buffer 10 mM dNTP 25 mM MgSO4 Primer Mix (10 µM each) Reverse primer Forward primer Template Applied Biosystem (ABI) Taq Autoclaved distilled water Total Quantity (µl) 5.0 PCR conditions 1) Initial denaturation: 94°C for 3 min 2) 35 cycles of a) DNA Denaturation: 94°C for 30 sec b) Primer annealing: 56.9 °C for 1 min c) DNA Extension: 68°C for 45 sec 1.0 4.0 2.0 2.0 2.0 0.5 3) Final extension: 72°C for 10mins 4) After cycling, the reaction is maintained at 18°C. 33.5 50.0 2.10 Bromo-uridine Triphosphate (Br-UTP) incorporation in permeabilized cells To label nascent RNA, intact cells were incubated in 7.5mM Br-uridine (Sigma #850187) for 3 h. Cells were rinsed with 1X PBS before being fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized in PBS/0.5% Triton X-100 for 5 min prior to immuno-staining. Incorporated Br-UTP was detected with anti-BrU antibody at 4°C overnight and incubated for at least 1 h with Alexa 596-conjugated goat anti mouse IgG at room temperature. 2.11 Micrococcal nuclease (MNase) accessibility assay MNase assays were performed as described by (Knoepfler et al., 2006). Briefly, U2OS cells were seeded in a 6 well-plate and subjected to scrambled or MLL5 siRNAs (#1 or #2+#3) for 72 h before harvest. The procedure for the MNase assay constituted five main steps: i) cell permeabilization, ii) MNase digestion of 55 permeabilized cells, iii) organic extractions of MNase digested DNA, iv) DNA precipitation and v) quantitation and assessment of DNA. The components of the buffers used are listed in Table 16. i) Cell Permeablization Unless otherwise stated, reaction was done at room temperature. To each well of a 6well plate, the medium from the cells was aspirated and 850µl room temperature (RT) permeabilization solution 1 (PS1) was added. After which, PS1 was removed and cells were treated with 0.025% lysolecithin (diluted from 1 mg/ml stock in 37◦C permeabilization solution 1 to 480µl total volume) at room temperature for 2 min. The solution was removed from the plate and 850µl of room temperature PS1 (without lysolecithin) was added. ii) MNase digestion of permeabilized cells After aspirating the ssolution from plate, 480µl of RT permeabilization solution 2 (PS2) containing 0, 6.25, 12.5, 25, 50 and 100 Units MNase respectively was added into each well and the solution was incubated for 5 min at RT. After which, the solution was discarded and 480µl 2× TNESK solution was added with gentle swirling to ensure complete cell lysis. Then, 480µl lysis dilution buffer was added and the resultant mixture was transferred to a 15-ml conical polypropylene tube. The tube was ◦ capped and incubated overnight at 37 C. 56 iii) Organic extractions of MNase digested DNA The cell lysate was diluted with 1 volume TE buffer, pH7.9 and 1 volume of neutralized phenol was added. The tube was inverted sharply several times before placing on a gentle shaker for 15 min. The samples were then centrifuged for 5 min at 2000 rpm. The upper aqueous layer was then transferred to a fresh tube. 1 volume of chloroform was added and the tube was inverted sharply several times before gently shaking for 15 min. The upper aqueous phase was obtained after centrifuging for 5 min at 2000 rpm. 1/10 volume of 3 M sodium acetate was then added and the mixture was mixed by inversion. iv) DNA precipitation 2.5 volumes of 95% ethanol was added to the above mixture and inverted gently for 20 times before incubating at −20◦C overnight. The next day, the mixture was centrifuged for 10 min at 10 000 X g and the supernatant was discarded. 0.5 ml of 70% ethanol was then added and gently inverted before centrifuging for another 2 min. After that, the supernatant was discarded and the pellet was dried for 5 min before the DNA was re-suspended in 100 µl TE buffer. v) Quantitation and assessment of DNA Finally, the concentration of the DNA was measured and 0.5 μg of each DNA sample was loaded onto a 1.2% agarose gel to assess the level of endogenous nuclease and MNase cleavage of the chromatin. 57 Table 16: Components of buffers used in MNase assay Buffers Permeabilization solution 1 (PS1) Permeabilization solution 2 (PS2) 2X TNESK Lysis dilution Components 150 mM sucrose 80 mM KCl 35 mM HEPES, pH 7.4 5 mMK2HPO4 5 mMMgCl2 0.5 mM CaCl2 150 mM sucrose 50 mM Tris·Cl, pH 7.5 50 mM NaCl 2 mM CaCl2 20 mM Tris·Cl, pH 7.4 0.2 M NaCl 2 mM EDTA 2% SDS 0.2 mg/ml proteinase K (add just before use) 150 mM NaCl 5 mM EDTA 58 CHAPTER 3 – RESULTS 3.1 Co-localization of MLL5 with the spliceosome components Our group has previously demonstrated that MLL5 forms intra-nuclear foci in interphase cells (Deng et al., 2004); however, its biological functions remain unclear. To investigate the possible biological processes involved, we first examined the costaining pattern of MLL5 with several well-characterized proteins that are known to display nuclear speckle pattern by immunofluorescence staining in HeLa cells. These proteins include CREB binding protein (CBP), kinetochore associated protein using CREST antibody, promyelocytic leukaemia (PML), spliceosome proteins like splicing factor SC35 and smith antigens (Sm). α-Sm antibody is directed against 7 proteins (B/B', D1, D2, D3, E, F, G) that constitute the common core of U1, U2, U4 and U5 small nuclear ribonucleoprotein particles (snRNP antigens) of the spliceosome complex. Surprisingly, MLL5 showed a high degree of co-localization with the spliceosome protein SC35 but not with the other proteins tested (Figure 5). Such colocalization was observed to be distributed in the nucleoplasm, excluding the nucleoli. 59 Figure 5: Co-localization of MLL5 with the spliceosome components. HeLa cells were co-stained with anti-MLL5-8009 antibody and other known proteins that displayed nuclear speckle pattern. These proteins include CREB binding protein (CBP), kinetochore associated protein using CREST antibody, promyelocytic leukaemia (PML), spliceosome proteins like smith antigens (Sm) or splicing factor SC35. MLL5 showed a close resemblance to nuclear speckles associated with the spliceosome complex, SC35 and Sm. Arrows indicated coiled bodies observed in the nucleoplasm when stained with anti-Sm antibody. These coiled bodies were not observed to co-localize with MLL5. Bar: 10 µm. 60 The above co-localization was observed using anti-MLL5 antibody (designated as αMLL5-8009) that recognised the central region of MLL5 (amino acids 1157–1170). We have also attempted to test the co-localization between MLL5 and SC35 with other self-generated anti-MLL5 antibodies that recognised other epitopes on MLL5 as well as commercially available anti-MLL5 antibodies. Anti-MLL5-227 antibody recognised amino acid residues 227-241 of full length MLL5. As shown in Figure 6, anti-MLL5-8009 antibody showed the most distinct MLL5 speckles and the extent of overlap between MLL5 and SC35 was the greatest. Therefore throughout this study, anti-MLL5-8009 antibody would be used to probe for full length MLL5 on Western Blot, immunoprecipitation and immunofluorescence. Faint or no signals (even at a low dilution factor of 1:25) were obtained with the other commercially available antibodies, except for a polyclonal anti-MLL5 antibody (Abcam #75339). These drastic differences in immunofluorescence signals could be attributed to the quality and specificities of various anti-MLL5 antibodies, where the specificities of the antibodies are largely dependent on the epitope to which the antibodies have been designed to recognise. In addition, these commercial antibodies also gave faint or no signals on Western Blot. To our knowledge, the crystal structure of MLL5 is still unknown; hence, it remains a challenge towards designing a good MLL5 antibody for a broad spectrum of applications. 61 Figure 6: Different anti-MLL5 antibodies and their co-localization with SC35. Comparing the different anti-MLL5 antibodies currently available, anti-MLL5-8009 antibody which recognised amino acid residues 1157 to 1170 of full length MLL5 showed the most distinct MLL5 speckles staining and the extent of overlap with SC35 was the greatest. Anti-MLL5-227 antibody recognised amino acid residues 227 to 241. Among the commercial antibodies, only a polyclonal anti-MLL5 antibody (Abcam #75339), showed the most promising immunofluorescence staining capabilities. However, the specific recognition site of this antibody on the N terminal of MLL5 is not disclosed on the product sheet. Bar: 10µM. To further verify the association with the spliceosome complex and to validate that the co-localization of MLL5 with the spliceosome components was not cell-type specific, we examined another spliceosome protein, smith antigens (Sm) along with SC35 in five human cell lines (293T, COS7, HeLa, U2OS, and WI38). Among these cell lines, 293T and COS7 are transformed cell lines; HeLa and U2OS are tumor cell lines while WI38 is a normal diploid fibroblast cell line. Anti-Sm antibody used was directed against seven proteins (B/B', D1, D2, D3, E, F, G) that constituted the 62 common core of U1, U2, U4 and U5 small nuclear ribonucleoprotein particles (snRNP antigens) of the spliceosome complex. Similar to SC35, the staining pattern of anti-MLL5-8009 antibody overlapped extensively with the snRNP antigens. The high degree of co-localization between MLL5 and spliceosome components was consistent across all the five cell lines tested (Figure 7). 63 Figure 7: Co-localization of MLL5 with the spliceosome components in different cell lines. Co-localization between MLL5 and the spliceosome components were consistent across five human cell lines (293T, COS7, HeLa, U2OS, and WI38). Among these cell lines, 293T and COS7 are transformed cell lines; HeLa and U2OS are tumor cell lines while WI38 is a normal diploid fibroblast cell line. Bar: 5 µm. 3.2 Localization of MLL5 and spliceosome components in response to nuclease and heat-shock treatment It has previously been demonstrated that SC35 and snRNP antigens co-localized within the interchromatin granule clusters (IGCs) and perichromatin fibrils (PFs) but their localizations within these nuclear structures displayed different nuclease sensitivities (Spector et al., 1991). This suggested that spliceosome components might localize through different molecular interactions. To determine the molecular basis 64 responsible for the association of MLL5 with the spliceosome components, the subnuclear distribution of MLL5 in response to heat-shock and RNase A treatments were examined and compared to that of SC35 and snRNPs in HeLa cells by immunofluorescence staining. As shown in Figure 8, cells digested with RNase A showed no alteration in the speckle morphology for both MLL5 and SC35. MLL5 and SC35 still co-localize extensively in RNase-A treated cells. However, RNase A-treated cells labelled with anti-Sm antibody not only showed an overall decrease in the fluorescence intensity of the snRNP antigens, these speckles also became diffusely distributed. These observations for snRNP antigens were consistent with previous report (Spector et al., 1991). These observations demonstrated that although MLL5 and the splicing components co-localized in the same nuclear speckle compartment, MLL5 evoked a response that preferentially resembled that of SC35 as compared to the snRNPs. Heat-shock treatment was previously performed in Drosophila (Yost and Lindquist, 1986) and mammalian cells (Bond, 1988) to inhibit pre-mRNA processing where U2 and U4/U5/U6 components were found to be disrupted in heat-shock treated cells (Bond, 1988; Shukla et al., 1990). Such treatment elicited a re-distribution and a decrease in the fluorescence intensity of the snRNP speckles except for the coiled bodies which remained visible in the nucleoplasm. Unlike snRNP speckles, SC35 speckles aggregated into rounded clusters and became enlarged with less evident interconnections (Spector et al., 1991). To determine the effect of heat-shock on MLL5, HeLa cells were heat-shocked for 15 min at 45°C before examining the cellular localizations of MLL5, SC35 and snRNP. Control cells were incubated for the 65 same duration in pre-warmed medium kept at 37°C. As seen in Figure 8, while the speckled pattern of MLL5 appeared evenly distributed in control cells; after heatshock, MLL5 speckles became less apparent. We speculate that the decreased speckle signals were likely due to the heat sensitivity of MLL5. Such heat sensitivity made the comparison for the co-localization pattern of MLL5 with SC35 or Sm in response to heat-shock treatment difficult. Nonetheless, previous RNase A treatment results might imply that MLL5 is functionally more related to SC35 as compared to the snRNP. 66 Figure 8: Association of MLL5 with splicing factor SC35 under RNase A digestion and heatshock. HeLa cells were treated with RNase A (100µg/ml) for 2 h. No change in the distribution pattern of MLL5 or SC35 under RNase A treatment was observed and both proteins were still co-localizing extensively. However, such treatment altered the speckled distribution of snRNP antigens as the speckles become significantly reduced and diffused throughout the nucleoplasm but the coiled bodies remain evident in the nucleoplasm. HeLa cells exposed to heat shock at 45°C for 15 min caused MLL5 speckles to become less evident as compared to the control cells. SC35 speckles not only become enlarged and rounded; the interconnections between the speckles also became less apparent. Instead of appearing as enlarged speckles, snRNP antigens appear to be uniformly distributed throughout the nucleoplasm excluding the nucleoli. Bar: 10µM. 3.3 Association of MLL5 and SC35 The high degree of co-localization between MLL5 and SC35 encouraged us to test if MLL5 physically interacted with SC35. Full-length MLL5 and its deletion fragments, MLL5-ΔCT (1-1150aa), MLL5-ΔPS (562-1858aa), MLL5-ΔCD (Δ562-1150aa) and MLL5-CD (562-1150aa) (Figure 9) tagged with FLAG epitope were co-transfected with hemagglutinin (HA)-tagged SC35 into 293T cells, followed by immunoprecipitation with anti-FLAG antibody. As shown in Figure 9, full-length MLL5 interacted with SC35 and the strongest affinity with SC35 was observed in the 67 deletion mutant that retained the central domain and the C-terminus (MLL5-ΔPS). Central domain alone (MLL5-CD) showed less affinity to SC35 as compared to MLL5-ΔPS, suggesting that the presence of C-terminal domain would enhance its association to SC35. Nonetheless, the key region responsible for the interaction was the central domain since the deletion mutant (MLL5-ΔCD), lacking the central domain displayed significantly reduced association with SC35. 68 Figure 9: Association of MLL5 with splicing factor SC35. (Top) A schematic representation of MLL5 and its deletion fragments, MLL5-ΔCT (1-1150aa), MLL5ΔPS (562-1858aa), MLL5-ΔCD (Δ562-1150aa) and MLL5-CD (562-1150aa) aa, amino acids. (Bottom) Full length MLL5 and various deletion mutants were immunoprecipitated (IP) from 293T cell lysates with anti-FLAG antibodies and detected by anti-HA or anti-FLAG antibodies. MLL5-CD is the key region responsible for the interaction with SC35. The numbers indicate the molecular masses (kDa) of the protein standards. WB, Western blot. CT, C terminus. CD, Central domain. PS, PHD SET domain. 69 3.4 Alteration in MLL5 protein level induced the re-distribution of SC35 to enlarged speckle domains It has been reported that the disassembly of inter-chromatin granule clusters as a result of transcription or pre-mRNA inhibition induced SR proteins such as SC35 to accumulate in enlarged nuclear speckles (Bregman et al., 1995). Since the localization of splicing factors in the nucleus has been demonstrated to be highly dynamic, we wanted to know if the physical association of MLL5 with SC35 was important for the dynamic structure of the speckle morphology. We began by examining the expression level and cellular localization of SC35 upon down-regulation of MLL5. U2OS cells were transfected with two different MLL5 siRNAs (MLL5-siRNA #1: targets to the coding sequence; MLL5-siRNA #2 and MLL5-siRNA #3: target to the 3’untranslated region) for 3 days before analysis by Western blotting or immunofluorescent staining. In this study, MLL5-siRNA #2 and MLL5-siRNA #3 are used in combination to obtain a knockdown efficiency that is comparable to MLL5-sRNA #1. As seen in Figure 10, no effects on SC35 protein expression were seen as compared to the negative control-siRNA (NC-siRNA) treated cells. 70 Figure 10: SC35 protein expression remains unaltered in MLL5 depleted cells. Total cell extract was prepared after U2OS cells were transfected with negative control (NC) or MLL5-siRNA (#1 or #2+#3) for 72 h. The expression of MLL5, SC35 and α-tubulin were studied by Western blotting. The expression of SC35 was not affected in MLL5-depleted cells as compared to NC-treated cells. Interestingly, knockdown of MLL5 resulted in enlarged, dot-like-SC35 speckles that lacked interconnections in contrast to NC-treated cells that displayed irregularly shaped SC35 speckles that appeared to be interconnected via a reticular network (Figure 11 Top). Phenotypes observed were scored through random selection of cells (n > 100 cells per sample) and categorised into 3 groups (Figure 11 Bottom): Normal (cells that exhibit irregular, punctate speckles with interconnections), Enlarged (cells that exhibit large spherical speckles without interconnections) and Others (cells that exhibit no speckles or a mixture of traits present in Normal and Enlarged groups). In NC-siRNA treated cells, 95% (COS7) and 96% (U2OS) exhibited typical SC35 speckle morphology (Normal group) while 5% (COS7) and 4% (U2OS) deviated 71 from this normal group (Enlarged + Others). In MLL5-siRNA #1 treated cells, there was an approximately 10-fold increase in the enlarged population: 55% (COS7) and 40% (U2OS) displayed enlarged SC35 speckles as compared to NC-siRNA-treated cells. More than 50% of MLL5-siRNA #1 treated cells displayed the enlarged phenotype as compared to NC-treated cells. 72 Figure 11: Alteration in MLL5 protein levels by RNA interference induced the re-distribution of SC35 to enlarged speckle domains. (Top) MLL5 depletion resulted in an enlargement of SC35 nuclear speckles with less evident interconnections as compared to the NC-siRNA treated cells in both cell lines, COS7 and U2OS. (Bottom) Phenotypes observed were scored through random selection of cells (n > 100 cells per sample) and categorized into 3 groups: Normal (cells that exhibit irregular, punctate speckles with interconnections); Enlarged (cells that exhibit large spherical speckles without interconnections) and Others (cells that exhibit no speckles or a mixture of traits present in Normal and Enlarged groups). 73 We also examined if ectopic over-expression of MLL5 would affect the cellular localization of SC35. 293T cells were transiently transfected with GFP-MLL5. After 48 h, cells were fixed and stained with anti-SC35 antibody. Similar to the results obtained from MLL5 knockdown, 90% of GFP-MLL5 positive cells displayed an enlargement of SC35 speckles; in contrast, only 1% of GFP-MLL5 negative cells exhibited enlarged SC35 speckles (Figure 12). Our findings suggest that changes in the protein homeostasis of MLL5 have an effect on the dynamic nuclear distribution of SC35. In addition, recent studies have suggested that SC35 is necessary to promote RNAPII elongation in a subset of genes and participate in the bi-directional coupling between transcription and splicing (Milne et al., 2005; Caslini et al., 2009). This raises a possibility that MLL5 may co-ordinate with SC35 to modulate the activity of these processes. 74 Figure 12: Exogenous introduction of MLL5 induced the re-distribution of SC35 to enlarged speckle domains. Ectopic over-expression of GFP-MLL5 in 293T cells resulted in an enlargement of SC35 speckles in GFP positive cells. Phenotypes observed in GFP negative and GFP positive cell populations were tabulated. More than 90% of GFP-positive cells displayed an enlargement of SC35 speckles as compared to GFP-negative cells. 75 3.5 Multiple transcription inhibitors induce MLL5 to redistribute to enlarged speckles Nuclear speckles are dynamic structures implicated in the spatial coordination of transcription and splicing (Misteli and Spector, 1999; Sacco-Bubulya and Spector, 2002). Such enlarged, dot-like SC35 speckles have previously been suggested to be an indication of altered splicing and/or transcription activity (O'Keefe et al., 1994). To examine the effect of transcription inhibition on the distribution of MLL5 speckles, we first employed several transcriptional inhibitors that exhibited different mechanism of actions, including α-amanitin (Bushnell et al., 2002), 5,6-dichloro-1-ßD-ribobenzimidazole (DRB) (Tamm et al., 1976; Chodosh et al., 1989), Actinomycin D (Perry and Kelley, 1970) and Roscovitine (Ljungman and Paulsen, 2001). As shown in Figure 13, in transcriptionally active nuclei (absence of inhibitors), the distribution pattern of MLL5 largely resembled that of SC35 and both MLL5 and SC35 speckles co-localized extensively. In transcriptionally inactive nuclei (presence of inhibitors), speckle morphology of both MLL5 and SC35 changed dramatically, from the normal irregularly shape to large rounded speckles without interconnections. Under each drug treatment, such enlargement of nuclear speckles observed for SC35 was consistent with previous studies showing a modification in the speckle morphology when cells were stimulated with transcription inhibitors (Lallena and Correas, 1997; Shopland et al., 2002). Altogether, the data suggest that MLL5 associates with SC35 in a specific nuclear compartment and is sensitive to the transcriptional state. Down-regulation of MLL5 with MLL5-siRNA, exogenous overexpression of GFP-MLL5 or the addition of transcription inhibitors led to an enlargement of nuclear speckles for both MLL5 and SC35. 76 Figure 13: Multiple transcription inhibitors induce MLL5 to redistribute to enlarge speckles. HeLa cells were treated with various transcription inhibitors at the respective final concentrations and duration: α-amanitin (10 µg/ml, 8 h), 5,6dichlorobenzimidazole riboside (DRB, 100 µM, 3 h), Actinomycin D (20 µg/ml, 2 h) (Sigma #A9415) and Roscovitine (25µM, 1.5 h). Control cells have irregularly shaped speckles with apparent interconnections while in transcription inhibited cells, speckles became rounded and enlarged lacking interconnections. Bar: 10µM 77 3.6 Intra-nuclear reorganization of MLL5 speckles is reversible and temperature dependent The transcriptional inactivator, DRB, is an adenosine analogue that suppresses RNAPII transcription by inhibiting the protein kinases that phosphorylate RNAPII CTD (Zandomeni and Weinmann, 1984; Stevens and Maupin, 1989). Cells incubated with DRB caused pre-mRNA splicing proteins to be re-distributed. Unlike α-amanitin that binds tightly and directly to RNAPII and inhibit transcription irreversibly, DRB can diffuse rapidly into the cell membrane and wash-out easily to reverse the transcriptional block (Tamm et al., 1976). Using DRB wash-out experiment, the redistribution of MLL5 and SC35 was found to be reversible and temperature dependent. After HeLa cells were treated with DRB for 3 h, both MLL5 and SC35 speckles were transformed from the usual irregular shaped speckle pattern to unconnected rounded dots as seen in Figure 14. After DRB wash-out, the cells were maintained at 37°C or 4°C for 1 h. For cells that were incubated at 37°C after the wash-out, MLL5 and SC35 speckles reverted back to the original pattern and colocalized extensively. Such phenotype was markedly different from those cells incubated at 4°C after the wash-out where MLL5 and SC35 speckles still remain enlarged without interconnections. This data complements our earlier observations that the speckle pattern of MLL5 correlates with the overall transcriptional activity of the cell. Both MLL5 and SC35 are likely to redistribute through a common mechanism that is energy-dependent. 78 Figure 14: Re-distribution of MLL5 speckles is temperature dependent. Intranuclear re-distribution of MLL5 speckles is reversible and temperature dependent. HeLa cells were initially treated with DRB at 100µM for 3 h. Thereafter, DRB medium was washout before fresh complete media was introduced. Each of the dishes was maintained at 37°C or 4°C for 1 h prior to fixation and labelled with anti-MLL5 and anti-SC35 antibodies. MLL5 and SC35 speckles remained enlarged in DRB washout and maintained at 4°C as opposed to the dish at 37°C. Bar: 10µM 3.7 Alteration in MLL5 expression triggered transcription block Next, we would like to examine whether knockdown of MLL5 may have an impact on the transcriptional efficiency of RNAPII. We assessed the activity of RNAPII by measuring the transcript levels of ribosomal subunit S14 which has been previously reported by Leuenroth and colleagues (Leuenroth and Crews, 2008) as an approach to monitor RNAPII activity. Cells were grown for 72 h to ensure efficient knockdown by the respective MLL5 siRNAs. MLL5-siRNA #2 and MLL5-siRNA #3 were used in 79 combination to achieve a knockdown efficiency that was comparable to MLL5siRNA #1. Subsequently trizol extraction was performed to extract the RNA for further analysis by semi-quantitative real time PCR (qPCR). As shown in Figure 15, ribosomal protein S14 transcript that was transcribed by RNAPII showed a significant decrease as compared to the NC –siRNA treated cells. Figure 15: Gene expression of S14 ribosomal subunit after MLL5 knockdown. U2OS cells were treated with the respective MLL5 siRNAs for 72 h before the cells were harvested for semi-quantitative real time PCR (qPCR). MLL5 siRNA #2 and MLL5 siRNA #3 were used in combination to achieve a knockdown efficiency that was comparable to MLL5 siRNA #1. As compared to the NC- siRNA treated cells, MLL5 siRNA treated cells showed a decrease in the ribosomal protein S14 transcript. 80 As an alternative method to demonstrate how MLL5 participates in transcription, we performed 5-bromouridine 5´-triphosphate (Br-UTP) incorporation experiment in MLL5-siRNA treated cells. Such Br-UTP incorporation experiment has been used by several groups to illustrate that RNAPII transcripts were predominantly distributed in several hundred foci throughout the nucleoplasm and to examine the sites of RNA synthesis in vivo (Jackson et al., 1993; Wansink et al., 1993). U2OS cells were treated with MLL5-siRNA(#1 or #2 + #3) or Actinomycin D, a known transcription inhibitor that suppressed transcription by intercalating with DNA and inhibiting RNA synthesis (Sobell, 1985). Prior to paraformaldehyde fixation, cells were treated with 7.5mM BrUTP for 3 h. In NC-siRNA treated cells, 96.5% were Br-UTP positive. Strikingly, in MLL5-depleted cells, more than 90% were Br-UTP negative (Figure 16). This observation was comparable to Actinomycin D-treated cells. Taken together, these results indicate that down-regulation of MLL5 blocked transcription. 81 Figure 16: Alteration in MLL5 expression by RNA interference triggers transcription block. (Left) Br-UTP incorporation was tabulated in the respective MLL5 siRNA-treated cells and Actinomycin D-treated cells. (Right) In the negative control (NC), cells are Br-UTP positive. BrUTP incorporation was abrogated in MLL5 knockdown cells. Actinomycin D, a known transcription inhibitor, was used as a positive control. 82 To study the effect of MLL5 up-regulation on transcription, ectopic over-expression of GFP-MLL5 was performed in 293T and Br-UTP incorporation was examined by immunofluorescence staining. The expression level of GFP-MLL5 was observed by the intensity of green fluorescence in cells that were successfully transfected (Figure 17, Top) and categorised into 3 groups (Figure 17, Bottom): Strongly expressed (GFPBright), Weakly expressed (GFPDim) and No expression (GFPNull). In GFPNull group, more than 90% were Br-UTP positive as opposed to the GFPBright group where more than 90% were Br-UTP negative (labelled with arrow in Figure 17). In GFPDim group, Br-UTP positive was only visible in about 30% of cell population (labelled with arrows in Figure 17) with the majority being Br-UTP negative. The expression level of GFP-MLL5 appeared to be co-relating negatively to the percentage of BrUTP that was being incorporated. These findings indicate that changes in MLL5 protein level either through over-expression or siRNA-mediated knockdown decreased transcription activity. Collectively, given the close association between MLL5 and the splicing components as seen earlier, this signifies an important role of MLL5 in co-transcriptional splicing and prompts us to examine its relationship with the transcription machinery, RNAPII. 83 GFP-MLL5 Br-UTP DAPI MERGE Figure 17: Exogenous introduction of MLL5 triggered transcription block. (Top) GFP-MLL5 was expressed to various extents as observed by the intensity of the green fluorescence in cells that were successfully transfected. (Bottom) Cells were categorised into three groups: Strongly expressed (GFPBright), Weakly expressed (GFPDim) and No expression (GFPNull). In GFPNull group, more than 90% were BrUTP positive. In GFPBright group, more than 90% were Br-UTP negative (labelled with arrow). In GFPDim group, Br-UTP positive was only visible in about 30% of cell population (labelled with arrowhead) with the majority being Br-UTP negative. 84 3.8 Association of MLL5 and RNAPII To begin with, we examined whether MLL5 exhibited a co-localization pattern with RNAPII by immunofluorescence staining. As shown in Figure 18, unlike the distinct speckle-like staining pattern of MLL5, RNAPII showed a characteristic granular distribution, most probably due to its organisation into transcription factories that harboured enzymes for RNA synthesis (Cook, 1999). Figure 18: Distribution pattern of MLL5 and RNAPII. Unlike the distinct punctate staining of MLL5 speckles, staining pattern of RNAPII using the respective antibodies was generally observed to be distributed uniformly throughout the cell nucleus. CTD4H8 recognised both phosphorylated and unphosphorylated RNAPII, H14 recognised phosphor-serine 5 of RNAPII while H5 recognised phosphor-serine 2 of RNAPII. There seemed to be a certain degree of co-localization between MLL5 and RNAPII. Bar: 10µM 85 To further assessed the possible interaction between MLL5 and RNAPII, FLAGMLL5 was over-expressed in 293T cells and the cell lysates were immunoprecipitated by anti-FLAG antibody and probed with various RNAPII antibodies. RNAPII has two physiologically important phosphorylation sites; Serine-5 and Serine-2 in the heptapeptide repeats (YSPTSPS) at RNAPII CTD. CTD4H8 antibody recognizes both the phosphorylated and non-phosphorylated form of RNAPII, H14 recognizes phosphor-Serine 5 while H5 recognizes phosphor-Serine 2. As shown in Figure 19, FLAG-MLL5 was able to co-immunoprecipitate with both phosphorylated forms of RNAPII, as detected by H5 and H14 antibodies. RNAPII phosphor-Serine 2 is involved in transcription elongation where phosphorylation is catalysed by P-TEFb complex comprising Cdk9-Cyclin T subunits. Previous finding reported Cyclin T1 to be enriched to a greater extent within the nuclear speckles as compared to its interacting kinase, Cdk9 and it functions to recruit other binding partners to the nuclear speckles (Herrmann and Mancini, 2001). Interestingly, we found that MLL5 co-immunoprecipitated Cyclin T1 but not Cdk9. The failure in detecting Cdk9 in MLL5 eluate was possibly due to the portion of Cyclin T1 that existed free of Cdk9 as the latter has been reported to be present in several other complexes that do not contain Cyclin T1 (Kass et al., 1997; Peng et al., 1998). Another interesting observation was that the band of Cyclin T1 in the FLAG-MLL5 eluate appeared to be migrating at a slower rate as compared to the band of Cyclin T1 in the input. It is of interest to determine the slower migrating band of Cyclin T1. Nonetheless, we speculate that MLL5 could tether to the transcriptional machinery and may participate in the transcriptional process with RNAPII. 86 Figure 19: Association of MLL5 and RNAPII. Immunoprecipitation study showed that MLL5 associates with both phosphorylated forms of RNAPII. CTD4H8 recognised both forms of RNAPII, H14 recognised phosphor-serine 5 of RNAPII and H5 recognised phosphor-serine 2 of RNAPII. Interestingly, MLL5 coimmunoprecipitated Cyclin T1 but not Cdk9, possibly due to the portion of Cyclin T11 that existed free of Cdk9. Also, the band of Cyclin T1 in the FLAG-MLL5 eluate seemed to be migrating at a slower rate as compared to the band of Cyclin T11 in the input. 3.9 MLL5 overexpression resulted in a slower migration of Cyclin T1 It is noted that the Cyclin T1 protein present in the FLAG-MLL5 eluate (Figure 19) appeared to migrate slower as compared to the input lysate. It has been reported that when cells were treated with transcriptional inhibitors, splicing activity was reduced and the nuclear speckles labelled with anti-SC35 antibody were observed to be fewer in number, enlarged and rounded (O'Keefe et al., 1994). In addition, Herrmann et al (Herrmann and Mancini, 2001) reported that apart from Cdk9, Cyclin T1 was also 87 found to coalesce into enlarged speckles that coincided with SC35 labelling when cells were treated with transcription inhibitors, Actinomycin D or DRB. We thus speculate that the slower migrating band of Cyclin T1 could be a result of transcription inhibition. To test this, we carried out immunoprecipitation and probed for Cyclin T1 in two different settings. In the first setting, HeLa cells were treated with transcription inhibitor, DRB, for 3 h before the cells were harvested and immunoprecipitated by anti-MLL5-8009 antibody. In the second setting, we transfected FLAG-MLL5 into 293T cells for 48 h before the cells were harvested and immunoprecipitated by anti-Cyclin T1 antibody. Interestingly, as shown in Figure 20, the slower migrating band of Cyclin T1 was not observed in DRB treated cells. Instead, it was observed in cells that were transfected with FLAG-MLL5. This suggested that the slower migration was not attributed solely to transcription inhibition, but more likely due to post-translational modifications such as acetylation or phosphorylation that could have occurred on Cyclin T1 in the event of transcription inhibition and triggered by the over-expression of MLL5 through an unknown mechanism. Alternatively, this observation suggested that MLL5 could have a higher binding affinity towards the modified form of Cyclin T1. This observation certainly opens up a promising new direction in studying transcription inhibition caused by MLL5. 88 Figure 20: MLL5 overexpression resulted in a slower migration of Cyclin T1. HeLa cells were treated with transcription inhibitor, DRB, for 3 h before the cells were harvested and immunoprecipitated (IP) by anti-MLL5-8009 antibody. In the parallel experiment, FLAG-MLL5 was transfected into 293T cells for 48 h before the cells were harvested and IP by anti-Cyclin T1 antibody. Interestingly, the slower migrating band of Cyclin T1 was not observed in DRB treated cells but in cells that were transfected with FLAG-MLL5. 3.10 MLL5 knockdown does not affect the phosphorylation state of RNAPII Since the phosphorylation state of RNAPII co-relates with the transcription status of the cell, we would like to examine if the transcription block induced by knockdown of MLL5 could alter the phosphorylation status of RNAPII. α-amanitin, a known transcription inhibitor that affects the general level of phosphorylated RNAPII, is used as a positive control (Lindell et al., 1970). Following exposure of U2OS cells to NCsiRNA, MLL5-siRNA (#1 or #2+#3) for 72 h, cells were harvested and the phosphorylation level of RNAPII was visualised by Western blotting using phosphorepitope specific antibodies. Immuno-blotting analysis in Figure 21 showed that the phosphorylation states of RNAPII remained largely unaffected in comparison to αamanitin-treated cells which resulted in a decrease in both the phosphorylated forms. This observation implied that MLL5-induced transcriptional inhibition might not 89 affect the initiation or elongation process in a conventional way but occurred by other yet to defined mechanisms. Since it is known that the chromatin structure may regulate the transcription activity (Paranjape et al., 1994), we speculate that MLL5siRNA treated cells may cause chromatin modifications, leading to the reduction in the transcription activity of RNAPII. Figure 21: MLL5 knockdown does not affect the phosphorylation state of RNAPII. MLL5-siRNA #1 or MLL5-siRNA #2+#3 treated samples did not show a significant change in the phosphorylation state of RNAPII as opposed to α-amanitintreated control samples. The transcription inhibitor, α-amanitin, is known to decrease the total level of RNAPII (8WG16) and phosphor signals for both Serine-5 (H14) and Serine-2 (H5) [IIa - hypo-phosphorylated band; IIo - hyper-phosphorylated band]. 90 3.11 MLL5 knockdown affects chromatin structure Cellular levels of histone signatures, mainly histone methylation or acetylation, were examined in cultured cell lines transfected with scrambled siRNA or MLL5-siRNA (#1 or #2+#3) for 72 h before the cell lysates were harvested for analysis of the chromatin. Antibodies specific to mono-, di and tri-methylated H3K4, H4 acetylation and H3K9 tri-methylation were used. As shown in Figure 22, H3K4 tri-methylation and histone H4 acetylation chromatin markers were dramatically reduced upon MLL5 knockdown. Less effect was seen for H3K4 di-methylation and no changes in the expression of H3K4 mono-methylation and H3K9 tri-methylation was detected. As such, we speculate that the global chromatin structure would be affected in MLL5siRNA (#1 or #2+#3) treated cells. To address this, micrococcal nuclease (MNase) accessibility assays was conducted. Briefly, cells were treated with MLL5 siRNA #1 or MLL5 siRNA #2+#3 for 72 h before the cells were permeabilized with lysolecithin and treated with increasing amounts of MNase (0, 6.25, 12.5, 25, 50 and 100 Units) before comparing the MNase cleavage pattern between the NC-siRNA treated cells and MLL5-depleted samples. As seen in Figure 23, we observed a significant decrease in the MNase sensitivity in MLL5-siRNA (#1 or #2+#3) treated cells as compared to NC-siRNA treated cells. More units of the MNase enzyme were required to cleave the same amount of DNA and this was likely due to the global chromatin structure being more compacted and hence less accessible to MNase. This observation is consistent with the histone modifications described above and support the notion that MLL5-depleted cells has a widespread influence on chromatin structure, resulting in a decrease in global transcription activity and the subsequent splicing process. 91 Figure 22: Analysis of chromatin modifications in MLL5 knockdown cells. Cultured cell lines were transfected with scrambled siRNA or MLL5-siRNA (#1 or #2+#3) for 72 h before the cell lysates were harvested and the cellular levels of histone signatures were probed in MLL5-depleted cell lysates. Antibodies specific to mono-, di and tri-methylated H3K4, H4 acetylation and H3K9 tri-methylation were used. Transcriptional markers for gene activation, H3K4 tri-methylation and histone H4 acetylation, were seen to largely decrease upon MLL5 knockdown in all the three cell lines tested. 92 Figure 23: Analysis of chromatin organization in MLL5 knockdown cells. (Bottom) Concentration-dependent MNase assay (0, 6.25, 12.5, 25, 50 and 100 Units of MNase) was performed to analyse the changes on the global chromatin folding. MLL5-depletion rendered the in vivo chromatin to be less accessible to micrococcal nuclease as more units of enzymes are required to digest the same amount of DNA. 3.12 MLL5 and chromatin remodelling complex Mammalian SWItch/Sucrose Non Fermentable (SWI/SNF) complex comprises of at least nine subunits, including one of the two alternative ATPase subunits, Brm or Brg1 that provides the source of energy for chromatin remodelling. To further understand how MLL5 could have affected the chromatin organization, we asked if MLL5 could also be associated with proteins implicated in chromatin remodelling such as the SWI/SNF-related chromatin-remodelling complex. U2OS cells were 93 treated with MLL5 siRNA (#1 or #2+#3) for 72 h before cell lysates were harvested for examining the expression profiles of the SWI/SNF subunits including Brm, Brg1 and Brm/Brg1 associated factor (Baf) - Baf155. As shown in Figure 24, MLL5 depletion resulted in a concomitant decrease in the expression levels of Brm and Baf155. Even though the functional consequences of the depletion in the SWI/SNF subunits remains elusive, this preliminary result further supports the notion that MLL5 participates in transcription regulation through its effects on the chromatin structure, as revealed by alterations in histone modifications and the expression level of chromatin remodelling subunits. In addition, it would also be interesting to determine if MLL5 regulates Brm and Baf155 at the transcriptional level. Figure 24: Effect of MLL5 knockdown on SWI/SNF protein complex. U2OS cells were treated with MLL5 siRNA (#1 or #2+#3) for 72 h before the cells were harvested and probed for the proteins with the indicated antibodies. SWI/SNF, a chromatin remodelling complex, contains either Brg1 or Brm as the ATPase catalytic core subunit and a set of Brg1/Brm-associated factors (BAF). MLL5 knockdown caused a decrease in Brm and BAF155. 94 3.13 MLL5 and splicing activity There is significant evidence to support the notion that transcriptional elongation and pre-mRNA splicing are linked within the cell either temporarily, spatially or functionally (Rain et al., 1998; Orphanides and Reinberg, 2002). Therefore, it is rational to speculate that the splicing activity may reduce upon knockdown of MLL5. A double-reporter splicing assay designed by Nasim and colleagues was employed to carry out the study (Nasim et al., 2002). pTN23 plasmid was a kind gift from M. T. Nasim and I. C. Eperon (University of Leicester, Leicester, United Kingdom). pTN23 plasmid contained a target intron introduced between two reporters, β-galactosidase luciferase. The upstream β-galactosidase reporter is expressed regardless of splicing. Upon splicing, the internal translation termination signal is removed and this causes the upstream β-galactosidase reporter to be in-frame with the downstream luciferase reporter. The downstream luciferase reporter is expressed after splicing and the ratio of luciferase activity to β-galactosidase activity is dependent on the proportion of transcripts that are spliced. The principle of this splicing assay is illustrated in Figure 25. Briefly, 293T cells were first knockdown with MLL5-siRNA (#1 or #2+#3) and SC35- siRNA #1+#2 for 24 h before pTN23 plasmid was introduced. After 48 h, cells were treated again with the respective siRNA to ensure that MLL5 level remained minimal at the point of harvest at 96 h. As seen from the Western blotting results in Figure 26, the knockdown efficiency for all siRNA were relatively efficient. MLL5siRNA (#1 or #2+#3) treated cells displayed a modest decrease in the splicing activity as revealed by an accumulation of the un-spliced transcripts. Luciferase assay was 95 also conducted and a similar trend of a decrease in splicing activity was observed and the significance is reflected by the p-value. The reduced level was comparable to that of SC35-siRNA treated cells. This data further supports the importance of maintaining a basal level of MLL5. The alteration in MLL5 level can trigger a cascade of cellular events that include an impediment to transcription and splicing processes. Figure 25: A test system for determining the splicing efficiency in mammalian cells. The splicing efficiency assay system is based on the reporter genes that encode for β-galactosidase (β – gal) and Luciferase (Luc). These reporter genes are fused inframe through recombinant fragments of the genes encoding adenovirus (Ad) and the skeletal muscle isoform (SK) of human tropomyosin. The recombinant fragment contains three in-frame translation stop signals (XXX) in the intronic region. Upon transfection into the mammalian cells, the pre-mRNA can be processed in either one of the ways. Firstly, with inefficient splicing, the RNA produced contains premature termination codons, resulting in β-galactosidase activity. Secondly, if there is efficient splicing, the translation stop signals are removed and this leads to the production of a fusion protein, generating both β-galactosidase and luciferase activity. [Adapted from (Nasim et al., 2002) ] 96 Figure 26: Analysis of splicing efficiency in MLL5 knockdown cells. pTN23 plasmid was co-transfected in MLL5-depleted cells. RNA was prepared and the amplification products were resolved in 2% agarose gel. Splicing products are shown on the right-hand side. Cell lysates was prepared to check for the knockdown efficiency. MLL5 knockdown cells showed a decrease in splicing efficiency. 97 CHAPTER 4 – DISCUSSION 4.1 An overview In this study, we demonstrated that MLL5 co-localized and physically interacted with splicing factor SC35. A change in MLL5 protein level either by ectopic overexpression or siRNAs-mediated knockdown resulted in enlarged SC35 speckles that are known to correlate with defects in co-transcriptional splicing. We showed that MLL5 is sensitive to the transcription state of the cell and associates with the transcription machinery. Perturbation in MLL5 expression level affects global transcription activity through histone modifications and chromatin remodelling. Here, we documented for the first time the functional importance of maintaining MLL5 homeostasis as disruptions to MLL5 expression level can consequently lead to deregulation of transcription and splicing processes. 4.2 Importance of maintaining MLL5 at a homeostatic level The regulation of gene expression in multi-cellular organisms forms the basis of celltype specificity and aberrantly expressed genes have profound effects on cellular functions and underscore the onset of many diseases. The transcription of proteincoding genes in eukaryotes is governed by RNAPII. As shown in Figure 27 (Top), the synthesis of nascent transcripts by RNAPII involves multiple processes that occur either sequentially or in parallel. The C-terminal domain of the large subunit of RNAPII (RNAPII CTD) is sequentially and extensively phosphorylated and dephosphorylated during different stages of transcription. During active transcription, 98 formation of an open complex between RNAPII and the DNA template allows for continuous progression and is a prerequisite for transcription initiation. During transcription initiation, Serine 5 residue of the RNAPII CTD gets phosphorylated by TFIIH, a protein complex that constitutes Cdk7 and Cyclin H. As RNAPII enters into the transcription elongation phase, Serine 2 residue of the RNAPII CTD gets phosphorylated by P-TEFb, a protein complex that constitutes Cdk9 and Cyclin T. It is noteworthy to know that even though Cdk9 has been shown to associate with three related members of the Cyclin T family, T1, T2a and T2b (Peng et al., 1998; Wei et al., 1998), Cyclin T1 is the predominant Cdk9 associated regulatory cyclin being examined to date. Therefore, Cdk9-Cyclin T1 subunits will be highlighted in our study. Apart from the Cyclin T family, Cdk9 has also been found to bind to cyclin K. However, the function of Cdk9-Cyclin K is less clear (Yu and Cortez, 2011). Recent findings suggest Cdk9-Cyclin K to be involved directly in the maintenance of the genome integrity. Moreover, the depletion of Cdk9 or its Cyclin K but not Cyclin T regulatory subunit not only impairs cell cycle recovery in response to replication stress, but also induces spontaneous DNA damage in replicating cells. CDK9-Cyclin K also interacts with ATR and other DNA damage response and DNA repair proteins (Yu et al., 2010). However, the underlying mechanisms for these still remain elusive. The RNAPII CTD physically interacts with a large number of proteins and can be portrayed as a “docking site” for factors required for different mRNA maturation events that occur concomitantly with transcript elongation. As nascent transcripts are generated by RNAPII, spliceosome formation occurs in parallel and this is a dynamic process with constant shuttling of proteins and RNA components during the splicing reaction (Kim et al., 2011). As such, nascent transcripts can be simultaneously 99 assembled into splicing complexes and undergo splicing. In this study, we have identified MLL5 as a novel protein that not only associated with the phosphorylated RNAPII CTD, but also with the serine/arginine-rich (SR) spliceosome protein family, splicing factor SC35. Therefore, residing in this region brings MLL5 into close proximity with the transcription and pre-mRNA processing machineries, thereby facilitating its assembly and recruitment to the transcription active sites to regulate gene expressions. Since the splicing factor SC35 has been reported to have an active role in transcription elongation (Milne et al., 2005; Caslini et al., 2009), we propose that MLL5 can function as a mediator for co-transcriptional splicing by utilising its association with the elongating RNAPII and SC35 to assist in the recognition and splicing of the newly synthesized mRNA. In human, CA150 (150kDa) has been reported to localize in nuclear speckles and interacts with both transcription elongating RNAPII, phospho-CTD RNAPII and splicing factors, SF1 (Goldstrohm et al., 2001). CA150 has been suggested to play a role in coupling transcription and splicing in vivo through the following two mechanisms – (1) Modification of the activity of protein-kinase complex that interacts with RNAPII; (2) Mediation of the recruitment of other effectors to the elongation complex (Goldstrohm et al., 2001; Sanchez-Alvarez et al., 2006). More work is needed in order to underlie the detailed molecular mechanism on how MLL5 participates in regulating the co-transcriptional splicing event. In addition, it is also crucial to identify other mediators that could have assisted MLL5 in the cross-talk between transcription and splicing. 100 The alteration of nuclear speckle morphology appears mostly dependent on MLL5 as inferred from the results obtained upon changing the level of MLL5. This is illustrated in the model in Figure 27 (Bottom). From our results, such alteration in MLL5 expression level remarkably reduced transcription activity through multiple ways. Histone signatures signifying active transcription, H3K4 tri-methylation and H4 acetylation, were significantly decreased and the chromatin became more compact. Even though the phosphorylation states of RNAPII remained unaffected, the closed complex between RNAPII and DNA template as a result of chromatin compaction impeded the progression of RNAPII. As such, no nascent RNA could be generated and this was revealed by the abrogation of Br-UTP incorporation. Besides, an accumulation of un-spliced transcripts through splicing assay was also observed, indicating a decrease in splicing activity. Based on these findings, we suggest that MLL5 could function as a gatekeeper in ensuring a smooth shuttling of the splicing factors and other accessory proteins between the various cellular compartments, in particular, the perichromatin fibrils and interchromatin granule clusters, to execute their functions in co-transcriptional splicing. Hence, in cells with altered level of MLL5, such trafficking is disrupted and this consequently led to a temporal aggregation of complexes which appeared as an enlarged-speckle phenotype, which has been associated with a disruption in transcription (Bregman et al., 1995) or splicing processes (O'Keefe et al., 1994). Cdk9/Cyclin T1 complex, also known as positive transcription elongation factor b (PTEFb), phosphorylates RNAPII CTD and this phosphorylation indicates the transition from transcription initiation to elongation. In the review by Cho et al (Cho et al., 2010), it has been suggested that post-translational modifications on the subunits of 101 the P-TEFb complex established a new link between modifications at the RNAPII complex, chromatin and the regulation of transcription elongation. These modifications include phosphorylation, ubiquitination, and acetylation. In fact, while phosphorylation and ubiquitination are common modifications shared with other Cdk/cyclin complexes, acetylation was first identified in Cdk9 and Cyclin T1. The slow migrating band of Cyclin T1 observed in Figure 20 could be a result of one of these modifications. We speculate that the altered levels of MLL5 could trigger a cascade of signaling process, which consequently led to post-translational modifications on Cyclin T1. This is highly probably as four acetylation sites (K380, K386, K390 and K404) have been identified in Cyclin T1. These sites are located in the highly structured predicted coiled-coil region of the Cyclin T1 and acetylation on these sites have been reported to negatively influence the binding properties between P-TEFb and 7SK small nuclear RNA (snRNA). Although the exact mechanisms on how acetylation disrupted the 7SK snRNA ribonucleoproteins complex and liberated P-TEFb remained unclear, it is striking that acetylated residues occur in those positions of the predicted coiled-coil structure are well positioned for protein-protein interactions (Cho et al., 2009). In the context of our study, future studies include determining the significance of MLL5 having a higher affinity towards the modified form of Cyclin T1. Perhaps, such modifications could be a critical tethering factor for MLL5 to associate with RNAPII during transcription elongation. Given the importance P-TEFb has towards the transcriptional activity of the cell, MLL5 could have a synergistic role with this complex and may play a crucial role in the global regulation during the transcription process. Therefore, unraveling the cause of the slow migrating band of Cyclin T1 due to changes in MLL5 level will certainly bring 102 important clues to the cross-talk between distinct protein modifications and the role of MLL5 during transcription. 103 Figure 27: A model illustrating the participation of MLL5 in transcription and splicing processes. (Top) In cells with basal level of MLL5, MLL5 interacts with the SR family proteins such as splicing factor SC35. P-TEFb complex phosphorylates at Serine-2 in the heptapeptide chain of the CTD while TFIIH phosphorylates at Serine5. Br-UTP is incorporated into nascent RNA as it is being synthesized by the spliceosome. H3K4 trimethylation and H4 acetylation denote transcription activation markers. Arrow indicates the direction of RNAPII moving along the transcription template. (Bottom) In cells with altered level of MLL5, MLL5 colluded into the enlarged speckle as denoted by the boundary together with other components, RNAPII and spliceosome. H3K4 trimethylation and H4 acetylation markers decrease and chromatin compaction occurs. This temporarily ceases the advancement of RNAPII along the transcription template. As a consequence, RNA synthesis is halted and Br-UTP incorporation is abrogated. See text for details. 104 4.3 Plausible roles of MLL5 transcription regulation 4.3.1 MLL5 and its involvement in histone modifications Trithorax protein family, in combination with other protein complexes, exert chromatin remodelling and histone-modifying activities that dictate cell fate and are indispensable for proliferation, development or differentiation (Schraets et al., 2003). Studies have demonstrated that MLL family contain diverse functional domains and global analysis of H3K4 methylation defines MLL family member targets. MLL1 has been the most extensive studied member and participates in a large Set1 complex that acts to maintain transcriptional activation states of target genes (Nakamura et al., 2002). It has been reported that Wdr82, a specific component of Human Set1/COMPASS, regulates H3K4 tri-methylation (Wu et al., 2008). RNA interference-mediated knockdown of Wdr82 resulted in a global reduction in H3K4 tri-methylation levels, with little to no effect on H3K4 mono- or di-methylation levels. The group suggested that the loss of Wdr82 could possibly affect the stability of the entire Set1A complex, a H3K4 methylase. Another histone modification that has been suggested to be strongly correlating with transcription activation in a wide variety of eukaryotic systems apart from H3K4 methylation (particularly, the tri-methylated state), is histone acetylation (Strahl et al., 1999; Santos-Rosa et al., 2003). In the study by Dou and colleagues, the group purified a stable complex (MLL1-WDR5) containing both MLL1 and the MYST family histone acetyltransferase MOF. This MLL1 complex was found to have MLL1-mediated histone methyltransferase activity that can affect mono-, di- and tri-methylation of H3K4 and a MOF-mediated histone 105 acetyltransferase activity that is specific for H4K16. MOF remodelled chromatin by histone acetylation and charge neutralization (Dou et al., 2005). The MLL1-MOF complex coordinately activated transcription of MLL1 target gene, HOXA9 gene expression in vitro. The knockdown of MOF caused a dramatic reduction of histone H4K16 acetylation which consequently down-regulated HOXA9 gene expression but not H3K4 methylation (Dou et al., 2005; Taipale et al., 2005). This indicated that H4K16 acetylation by MOF is dependent upon MLL1 but H3K4 methylation by MLL1 can occur independently of MOF. Nonetheless, both H3K4 methylation and H4K16 acetyltransferase activities were required for the optimal transcription activation of the MLL1 target HOXA9 gene. Similar to Wdr82 and MLL1-MOF complex, our results in Figure 22 showed different combinations of histone modifications that dictated the transcriptional responses and cellular functions upon changing MLL5 level .Whether these transcription activation markers stems from the intrinsic enzymatic activity of MLL5 remains to be delineated. It would thus be intriguing to investigate if MLL5 is a component of any histone modifying enzyme complex and this could be done using proteomic approaches or through mass spectrometry. Unravelling the putative MLL5-associated complexes would certainly aid in better understanding of how MLL5 regulate the transcription of its target genes. Nonetheless, in the work described by Sebastian and colleagues (Sebastian et al., 2009), MLL5 indirectly regulated H3K4 methylation by regulating the expression of histone-modifying enzymes LSD1 and SET7. 106 4.3.2 MLL5 and its involvement in chromatin organisation A condensation of the general chromatin structure in MLL5-siRNA treated cells prompted us to understand how MLL5 could exhibit such chromatin organisation ability. We speculate that the disruption of MLL5 homeostasis could destabilize the architectural scaffold for RNAPII. This could affect the genomic transcription template by altering the functions of transcription factors and chromatin remodelling enzymes. Such transcription stress could temporarily cease the advancement of the elongating RNAPII along the chromatin, thus inhibiting nascent RNA generation and reduced splicing activity. In the work described by (Knoepfler et al., 2006), myc proto-oncogenes were required for the widespread maintenance of active chromatin. To address whether myc levels influenced chromatin structure, the group conducted similar MNase accessibility assays using Tet-Off Myc B (P493-6) cell system (Schuhmacher et al., 1999) in which Myc could be reproducibly turned off by the addition of tetracycline. Results showed that both the loss and gain of Myc function substantially influenced widespread histone modifications. Similar to MLL5, down-regulation of Myc expression led to a decreased in active chromatin markers and DNA accessibility. It has been proposed that Myc may influence the global chromatin structure directly through the widespread binding of Myc to genomic DNA coupled with the recruitment of chromatin-modifying proteins; or indirectly through the up-regulation of the histone acetyltransferase GCN5. Since MLL5 does not have DNA binding motifs, we speculate that MLL5 exert its chromatin re-modification properties through an 107 indirect mechanism, such as by forming a bridging complex with the chromatinmodifying proteins. In the case of human transcriptional co-activator PC4, PC4-mediated chromatin condensation lies in the direct interaction with the core histones H3 and H2B where it functions to link the different widely separated nucleosomes. Hence, unlike MLL5 and Myc, silencing of PC4 resulted in chromatin de-compaction as evidenced by the increase in MNase accessibility (Das et al., 2006). Therefore, to unravel the biochemical mechanisms of MLL5 mediated transcriptional regulation on a global scale, future studies include defining the functional correlation of MLL5 with the histones, non-histone chromatin proteins (such as HP1, HMGs, and PARP-1) as well as chromatin modifying proteins (such as SWI/SNF complex). Even though preliminary study indicates that MLL5 depletion results in a concomitant decrease in Brm but not Brg1, the consequences of this decrease with respect to MLL5’s role in chromatin remodelling remains elusive. Study by Batsche and colleagues (Batsche et al., 2006) showed that Brm is a regulator of alternative splicing of several genes which include including E-cadherin, BIM, cyclin D1 and CD44. Brm also associates with several components of the spliceosome. To a certain extent, Brm is responsible for the crosstalk between transcription and RNA processing by decreasing RNAPII elongation rate and facilitating the recruitment of the splicing machinery to variant exons with suboptimal splice sites. Therefore, it would certainly be exciting to further address the potential synergistic role of MLL5 and Brm as transcription regulators. 108 CHAPTER 5 – FUTURE DIRECTION AND CONCLUSION 5.1 Chromatin remodelling, histone modifications, and DNA methylation - How does it all fit together? In this study, the data presented in this report demonstrate that changes in MLL5 expression influence transcription regulation, possibly through histone modifications and chromatin remodelling. Recent studies have also suggested a link between DNA methylation and transcription repression (Kass et al., 1997; Curradi et al., 2002; Flintoft, 2010). Such epigenetic modifications interfere with the binding of transcriptional machinery by changing recognition sites that involve cytosine, specifically at the CpG rich sites. CpG methylation facilitates the assembly of transcription repressor complexes that contain histone deacetylases, histone methylases and ATPase complexes that also mediate chromatin remodelling. The schematic diagram in Figure 28 summarizes the multiple layers of epigenetic modifications in the control of chromatin structure and gene expression. In this regard, it would be interesting to determine if MLL5 also affects DNA methylation so as to further elucidate the complex interplay between these various epigenetic mechanisms. Recently, Heuser and colleagues reported that the treatment of homozygous Mll5 lossof-function mice with a DNA de-methylating agent, 5-Aza-2’-deoxycytidine, led to a complete loss of repopulation activity, accumulation of hematopoietic progenitors and 109 a dramatic increase of mature cells in the bone marrow (Heuser et al., 2009). The treatment with a histone deacetylases (HDAC) inhibitor, Trichostatin A, did not show similar effects, suggesting that the observations were specific for DNA demethylation. Therefore, it is likely that Mll5 could regulate hematopoietic differentiation and/or hematopoietic stem cell (HSC) renewal through yet to known mechanisms that involve the initiation and/or maintenance of DNA methylation. These findings suggest that MLL5 could be a member of the chromatin associated proteins that influences CpG methylation regulated gene expression and is required for the maintenance of methylation at critical stages of haematopoiesis. Currently, chromatin immunoprecipitation (ChIP) grade anti-MLL5 antibodies are unavailable, thereby inhibiting the use of direct ChIP strategies to analyze the promoters of target genes that could possibly be affected as result of chromatin modifications. Therefore, it remains a challenge to link potential target genes to posttranscriptional regulatory programmes so as to reveal the physiological implications of MLL5. 110 Figure 28: Possible epigenetic modifications on the chromatin. A propose model for how multiple epigenetic modifications can convert unmethylated, ‘‘open’’ chromatin into methylated, ‘‘closed’’ chromatin. Arrows indicate the possible sites of each epigenetic action. HP1, heterochromatin protein 1; MBD, methyl-CpG-binding domain protein. [Adapted from (Geiman and Robertson, 2002) ] 5.2 Histone modifying properties of MLL5 – When does it occur? The histone methyltransferase (HMT) activity for MLL family proteins, except MLL5, has been extensively studied over the years. Till date, it remains elusive if MLL5 also possesses such intrinsic histone methyltransferase activity. Even though it has been reported that MLL5 suppressed the expression of Cyclin A2 via indirect regulation of H3K4 methylation through LSD1 and SET7/9 in quiescent myoblasts, no histone methyltransferase activity was detected in recombinant MLL5 in the in vitro system (Sebastian et al., 2009). However, it is noteworthy to highlight that the enzymatic activity of MLL5 can be achieved upon specific post-translational modifications. During mitosis, upon being phosphorylated by CDC2, this led to the re-localization of 111 MLL5 into cytoplasm, rendering the chromatin to be inaccessible to MLL5. Interestingly, another group discovered that a shorter form of MLL5, MLL5α (609 amino acids) could exert H3K4 histone methyltransferase activity only after the Thr440 residue of the SET domain is being GlcNAcylated (Fujiki et al., 2009). Therefore, it is plausible that MLL5 has to be post-translationally modified before it can exert its enzymatic properties. Yew and colleagues (Yew et al., 2011) also identified another isoform of MLL5, MLL5β (503 amino acids), that associated with transcription factor AP-1 at the distal region of the HPV18 long control region which consequently led to the activation of E6/E7 transcription. The SET domain in MLL5β was found to be responsible for the activation as inactivation of the SET domain decreased E6/E7 gene activation, though not depleting it completely. Nonetheless, this finding suggests that MLL5β may interact with other proteins apart from AP-1 to exert play a cooperative role for E6/E7 activation. In addition, other unidentified post-translational regulation on MLL5β could also be required for this activation. 5.3 Cell cycle arrest or transcription inhibition – Which comes first? It has been an age-old riddle that has perplexed generations: Which came first, the chicken or the egg? At present, our current model also poses a scientific and philosophical mystery, the interesting question on the correlation between transcription regulation and cell cycle progression. Previous findings by our group have showed that either knockdown or over-expression of MLL5 caused cell cycle arrest at G1/S or G2/M boundaries (Cheng et al., 2008). Therefore, does transcription inhibition causes cell cycle arrest or cell cycle arrest causes transcription inhibition? 112 Currently, there is no evidence on which phenomenon occurs first since transcriptional regulation changes continuously during cell cycle progression. For example, when chromosomes condensed into compact structures at M-phase, most factors required for active gene expression are inaccessible to the binding sites on DNA and cells undergo global transcriptional inhibition (Kang et al., 2008). In proliferating cells, cell cycle-dependent transcriptional regulation occurs simultaneously. Hence, it remains a challenge to resolve the cell cycle mediated effects of transcription inhibition or the reciprocal experimentally. 5.4 Conclusion In conclusion, this study serves as a preliminary investigation for the involvement of MLL5 in transcription regulation. However, the detailed molecular mechanisms remain unclear. At present, we have demonstrated that MLL5 is a novel interacting partner of SC35, where the latter has recently been reported to possess bridging capability between transcription and splicing processes. Given the high degree of colocalization observed between MLL5 and SC35, it is promising to investigate the synergistic effect of these two proteins in the co-transcriptional splicing process. Our results are also suggestive for the role of MLL5 in regulating transcription activity. An alteration in MLL5 level is observed to substantially influence transcription, possibly through a cascade of events that include histone modifications and chromatin remodelling. As the function of MLL5 in the context of global gene regulation is far from being entirely understood, a major goal for the future studies is to unravel the mechanisms downstream of MLL5 and identify potential in vivo 113 transcriptional targets. An attempt to elucidate if MLL5 possesses intrinsic histone methyltransferase activity will certainly open up a promising new direction for the role of MLL5 in epigenetic regulation. Investigations into this complexity will provide an insight into the fundamentals of gene regulation and chromatin organisation. This will greatly enhance the mechanistic understanding of MLL5 in transcription regulation, which is one of the major cellular events that define the cell fate. 114 REEFERENCES Aasland, R., T.J. Gibson, and A.F. Stewart. 1995. The PHD finger: implications for chromatinmediated transcriptional regulation. Trends Biochem Sci. 20:56-59. Ansari, K.I., B.P. Mishra, and S.S. Mandal. 2008. 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MLL5 in transcription regulation, thereby contributing to gene regulation and cell cycle progression Maintaining a proper intracellular balance of 35 MLL5 will also be important in providing a framework for proper cellular development as marginal alterations could serve as a determinant for the onset of diseases Most importantly, elucidating the transcriptional and splicing regulation not only enable... threonines Among them, modifications pertaining to active transcription include acetylation of histone 3 and histone 4 (H3 and H4) or dior tri-methylation of H3K4; and these are classified as euchromatin modifications Heterochromatin modifications are associated with inactive transcription, and methylation occurs on H3K9 or H3K27 These histone modifications consequently 23 cause a change in the net charge of. .. interactions with Serine -5 phosphorylated CTD to catalyse the co-transcriptional capping reaction Various splicing factors are recruited during the elongation phase of transcription to facilitate co-transcriptional splicing These splicing factors are dependent on Serine-2 phosphorylation on the CTD The 3’-end formation is functionally coupled to transcription termination Importantly, increasing evidence now... eviction In this study, we will examine how histone modifications and chromatin remodelling affect transcription Histone tails are susceptible to numerous post-translational modifications (Li et al., 2007) These modifications include methylation of arginine (R) residues; methylation, acetylation, ubiquitination, ADP-ribosylation, and sumoylation of lysines (K); and phosphorylation of serines and threonines... that the transcription and RNA processing machineries are functionally integrated in a reciprocal fashion such that individual co-transcriptional processing events can influence transcription at different phases [Adapted from (Pandit et al., 2008)] Recently, Lin and colleagues (Caslini et al., 2009) has uncovered a new and important role in transcription for a splicing regulator protein, SC 35, that... observed that RNAPII was accumulated within the gene body upon SC 35 depletion, indicating RNAPII stalling before it reached the end of the gene This stalling led to a decrease in RNAPII elongation, which was confirmed by measuring the nascent transcripts using a run -on assay that utilized non-radioactive nucleotides In short, these findings confirm the involvement of SC 35 in the bi-directional coupling between... transcription inhibition These observations suggest the role of MLL5 in the transcription or splicing process Given the close interplay between the transcription and splicing processes, the effects of changes in MLL5 expression level on transcription and splicing were examined MLL5 formed aggregates and localized in enlarged nuclear speckles in respond to various transcription inhibitors Br-UTP incorporation... at Serine2 (Ser2), stimulates transcriptional elongation In parallel, high elongation rates allow the simultaneous presentation to the splicing machinery of strong and suboptimal 3’ splice sites, which favours the use of the stronger one, leading to skipping of an alternative exon [Adapted from (Fededa and Kornblihtt, 2008)] 22 In summary, the continuous shuttling of splicing factors to active transcription. .. Emerging evidence has proved that functional integration of transcription by RNAPII and RNA processing machineries are mutually beneficial for efficient and regulated gene expression The transcription process progresses from the initiation phase to the elongation phase and finally, the termination phase and these coordinated events within the cell nucleus are briefly summarized in Figure 2 Research... speckles are dynamic structures and there is a continuous shuttling of splicing factors in and out of the speckles In the event of transcription inhibition, either through the use of inhibitors or as a consequence of heat-shock, nuclear speckles became enlarged and rounded as splicing factors aggregate in them (Spector et al., 1991; Melcak et al., 2000) However, when the expression of intron-containing ... Impact of altering the basal level of Mixed Lineage Leukemia on global chromatin organization and transcription regulation (Manuscript to be submitted) 10 SUMMARY Mixed Lineage Leukaemia (MLL5)... Antisense 5 -ACGUCACACGUUCGGAGAAdTdT-3’ Sense 5 -CGCCGGAAAAGGGAAAAUAdTdT-3’ Antisense 5 -UAUUUUCCCUUUUCCGGCGdTdT-3’ Sense 5 - CAGCCCUCUGCAAACUUUCAGAAUUdTdT-3’ Antisense 5 -AAUUCUGAAAGUUUGCAGAGGGCUGdTdT3’... suggest a novel cellular role of MLL5 in transcription regulation, thereby contributing to gene regulation and cell cycle progression Maintaining a proper intracellular balance of 35 MLL5 will also